Use of Semaphorin-4D Binding Molecules for Modulation of Blood Brain Barrier Permeability

ABSTRACT

Provided herein are methods for decreasing blood-brain barrier permeability in a subject with a neuroinflammatory disorder, comprising administering to the subject an effective amount of an isolated binding molecule which specifically binds to semaphorin-4D (SEMA4D) or to its high affinity Plexin-B1 receptor.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority benefit to U.S. Provisional Appl. No.61/545,809, filed on Oct. 11, 2011, U.S. Provisional Appl. No.61/555,726, filed on Nov. 4, 2011, and U.S. Provisional Appl. No.61/593,641, filed on Feb. 1, 2012, the contents of which are herebyincorporated by reference in their entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The content of the electronically submitted sequence listing in ASCIItext file (Name: “1843_(—)0680003_SequenceListing_ascii.txt”; Size:33,807 bytes; and Date of Creation: Oct. 10, 2012) filed with theapplication is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Semaphorin 4D (SEMA4D), also known as CD100, is a transmembrane protein(e.g., SEQ ID NO: 1 (human); SEQ ID NO: 2 (murine)) that belongs to thesemaphorin gene family. SEMA4D is expressed on the cell surface as ahomodimer, but upon cell activation SEMA4D can be released from the cellsurface via proteolytic cleavage to generate sSEMA4D, a soluble form ofthe protein, which is also biologically active. See Suzaki et al.,Nature Rev. Immunol. 3:159-167 (2003); Kikutani et al., Nature Immunol.9:17-23 (2008).

SEMA4D is expressed at high levels in lymphoid organs, including thespleen, thymus, and lymph nodes, and in non-lymphoid organs, such as thebrain, heart, and kidney. In lymphoid organs, SEMA4D is abundantlyexpressed on resting T cells but only weakly expressed on resting Bcells and antigen-presenting cells (APCs), such as dendritic cells(DCs). Its expression, however, is upregulated in these cells followingactivation by various immunological stimuli. The release of solubleSEMA4D from immune cells is also increased by cell activation.

SEMA4D has been implicated in the development of neurodegenerativediseases, autoimmune diseases, demyelinating diseases, and certaincancers. While the role of SEMA4D signaling through its receptors, e.g.,Plexin-B1, on angiogenesis is well-recognized, the effect of SEMA4Dsignaling on Blood Brain Barrier (BBB) remains unclear. This isimportant because changes in the permeability of the BBB have a profoundinfluence on brain tissue and function. There remains, therefore, a needfor treatments for neuroinflammatory disorders that arise as a result ofbreakdown in the BBB, and, in particular, therapeutics that inhibit,suppress, prevent, reverse, or slow the breakdown of the BBB.

BRIEF SUMMARY OF THE INVENTION

Methods for using semaphorin-4d binding molecules for modulation ofblood brain barrier permeability are disclosed herein. Evidence ispresented demonstrating that SEAM4D can compromise the integrity of theBBB thereby increasing its permeability. According to aspects of theinvention illustrated herein, there is provided a method for decreasingblood brain barrier permeability in a subject having a neuroinflammatorydisorder including administering to the subject an effective amount ofan isolated binding molecule which specifically binds to semaphorin-4D(SEMA4D), thereby decreasing blood brain barrier permeability in thesubject.

According to aspects illustrated herein, there is provided a method ofmaintaining or increasing Claudin-5 expression in a subject having aneuroinflammatory disorder comprising administering to the subject aneffective amount of an isolated binding molecule which specificallybinds to semaphorin-4D (SEMA4D), wherein the binding molecule maintainsor increases Claudin-5 expression in the subject.

According to aspects illustrated herein, there is provided a method ofdecreasing blood brain barrier permeability in a subject having aneuroinflammatory disorder, comprising administering to the subject aneffective amount of an isolated binding molecule which specificallyinhibits semaphorin 4D (SEMA4D) interaction with a SEMA4D receptorthereby decreasing blood brain barrier permeability in the subject.

According to aspects illustrated herein, there is provided a method oftreating a subject having a neuroinflammatory disorder, comprisingadministering to the subject an effective amount of an isolated bindingmolecule which specifically inhibits semaphorin 4D (SEMA4D) interactionwith a SEMA4D receptor, wherein the binding molecule decreasespermeability of the blood-brain barrier, thereby treating the subject.

According to aspects illustrated herein, there is provided a method ofdecreasing blood-brain barrier permeability in a subject having aneuroinflammatory disorder, comprising administering to the subject aneffective amount of an isolated binding molecule which specificallybinds to SEMA4D, wherein the binding molecule competitively inhibits areference monoclonal antibody selected from the group consisting ofVX15/2503 or 67 from specifically binding to SEMA4D.

According to aspects illustrated herein, there is provided a method oftreating a subject having a neuroinflammatory disorder, comprisingadministering to the subject an effective amount of an isolated bindingmolecule which specifically binds to semaphorin-4D (SEMA4D) and anisolated binding molecule which specifically binds to Plexin-B1, whereinthe SEMA4D and Plexin-B1 binding molecules decrease permeability of theblood-brain barrier, thereby treating the subject.

According to aspects illustrated herein, there is provided a method oftreating a subject having a neuroinflammatory disorder, comprisingadministering to the subject an effective amount of an inhibitor ofsemaphorin 4D (SEMA4D) interaction with a SEMA4D receptor, wherein theinhibitor decreases permeability of the blood-brain barrier, therebytreating the subject.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1: Schematic of the dynamic in vitro BBB (“DIV-BBB”) experimentalprotocol described in the Examples.

FIG. 2: In vitro DIV-BBB model showing measurements of BBB integrity asreflected in transendothelial electrical resistance (TEER) in thepresence of recombinant SEMA4D (0.05, 0.5, 5 or 50 μg/mL) and VX15/2503Antibody (“VX15”).

FIG. 3: In vitro DIV-BBB model showing measurements of BBB integrity asreflected in transendothelial electrical resistance (TEER) during theformation of the BBB, the breakdown of BBB in the presence ofrecombinant SEMA4D (0.5, 5 or 50 μg/mL), and the restoration of the BBBin presence of VX15/2503 Antibody (“VX15”), but not isotype control(“Iso”).

FIG. 4: In vitro DIV-BBB model showing measurements of BBB integrity asreflected in transendothelial electrical resistance (TEER) during theformation of the BBB, the breakdown of BBB in the presence of 0.25, 2.5,or 25 μg/mL of control C35 antigen (“CTRL”) or 50 μg/mL of recombinantSEMA4D, and the restoration of the BBB in presence of VX15/2503 Antibody(“VX15”).

FIG. 5: In vitro DIV-BBB model showing measurements of BBB integrity asreflected in transendothelial electrical resistance (TEER) during theformation of the BBB, the breakdown of BBB in the presence ofrecombinant SEMA4D (50 μg/mL), and the restoration of the BBB in thepresence of VX15/2503 Antibody (“VX15”), anti-Plexin-B1 antibody(“Anti-PLXNB1”), but not isotype control (“Iso”).

FIG. 6: In vitro DIV-BBB model showing measurements of BBB integrity asreflected in transendothelial electrical resistance (TEER) during theformation of the BBB, the breakdown of BBB in the presence of activatedPBMC (10⁶/ml) and flow cessation, and the restoration of the BBB in thepresence of VX15/2503 Antibody or Isotype Control IgG.

FIG. 7A-C: Results from the in vivo EAE model showing integrity of theBBB or loss thereof as reflected by immunostaining of fibrinogen(“Fib.+”) penetration into brain tissue (7A left panel and quantitationin 7B) and Claudin-5 (“CLN5+”) expression as detected by red stain (7Aright panel and quantitation in 7C) following treatment with VX15/2503antibody (“Anti-SEMA4D”) or isotype control (“Control IgG”).

FIG. 8: Immunoblot results showing the effect of increasingconcentrations of recombinant SEMA4D (1 ng/ml, 10 ng/ml and 100 ng/ml)on the expression of the key endothelial tight junction proteinClaudin-5 (“CLN-5”) compared to VEGF-A positive control in primary mousecentral nervous system (CNS) endothelial cultures.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

It is to be noted that the term “a” or “an” entity refers to one or moreof that entity; for example, “an anti-SEMA4D antibody” is understood torepresent one or more anti-SEMA4D antibodies. As such, the terms “a” (or“an”), “one or more,” and “at least one” can be used interchangeablyherein.

It should be noted that the term “blood brain barrier” and “BBB” areused interchangeably.

As used herein, the term “breakdown” or “disruption” with regards to theBBB, such as “blood brain barrier breakdown”, “blood brain barrierdisruption”, “breakdown of the blood brain barrier”, or “disruption ofthe blood brain barrier” refers to an increase in permeability of theblood brain barrier, or, in the case of the “DIV-BBB,” a humanizeddynamic in vitro model of BBB, a decrease in transendothelial electricalresistance (TEER). McCallister et al., Brain Res. 904:20-30 (2001);Santaguida et al., Brain Res. 1109:1-13 (200); and Cucullo et al.,Epilepsia 48:505-16 (2007) have shown that there is a direct (inverse)relationship between TEER and permeability in DIV-BBB. In addition, anincrease in the permeability of the blood brain barrier or a decrease inelectrical resistance can be the result of a decrease in the number,density and/or concentration of endothelial cells present on the BBB; ora change in the morphology or interactions among endothelial cells orastrocytes or between endothelial cells and astrocytes that form theBBB.

As used herein, the term “restoration” with regards to the BBB, such as“blood brain barrier restoration” or “restoration of the blood brainbarrier” refers to a decrease in permeability of the blood brainbarrier, or, in the case of the DIV-BBB, a humanized dynamic in vitromodel of BBB, an increase in transendothelial electrical resistance.

As used herein, the term “neuroinflammatory disorder” refers to acentral nervous system (CNS) inflammatory disorder, a neurodegenerativedisorder, an autoimmune disorder of the central nervous system, a myelindisorder or a disease that affects oligodendrocytes, or a post-traumamyelin disorder of the central nervous system. It should be noted thatneuroinflammatory disorders are often also neurodegenerative disorders.However, it is possible for a neurodegenerative disorder to exist in theabsence of obvious neuroinflammation. This is the case, for example, inlate stage secondary progressive multiple sclerosis.

The term “therapeutically effective amount” refers to an amount of anantibody, polypeptide, polynucleotide, small organic molecule, or otherdrag effective to “treat” a disease or disorder in a subject or mammal.In the case of a neuroinflammatory disorder, the therapeuticallyeffective amount of the drug can decrease the permeability of the BBB;reduce, retard or stop an increase in BBB permeability; inhibit, e.g.,suppress, retard, prevent, stop, or reverse an increased permeability ofthe BBB; increase the number, density and/or concentration ofendothelial cells present on the BBB; change in the morphology orfunction of endothelial cells; or a change in the interactions amongendothelial cells or astrocytes or between endothelial cells andastrocytes that form the BBB; relieve to some extent one or more of thesymptoms associated with an increased BBB permeability, e.g.,neuroinflammatory disorders; reduce morbidity and mortality; improvequality of life; or a combination of such effects.

Terms such as “treating” or “treatment” or “to treat” or “alleviating”or “to alleviate” refer to both 1) therapeutic measures that cure, slowdown, lessen symptoms of, reverse, and/or halt progression of adiagnosed pathologic condition or disorder and 2) prophylactic orpreventative measures that prevent and/or slow the development of atargeted pathologic condition or disorder. Thus those in need oftreatment include those already with the disorder; those prone to havethe disorder; and those in whom the disorder is to be prevented.Beneficial or desired clinical results include, but are not limited to,alleviation of symptoms, diminishment of extent of disease, stabilized(i.e., not worsening) state of disease, delay or slowing of diseaseprogression, amelioration or palliation of the disease state, andremission (whether partial or total), whether detectable orundetectable. “Treatment” can also mean prolonging survival as comparedto expected survival if not receiving treatment. Those in need oftreatment include those already with the condition or disorder as wellas those prone to have the condition or disorder or those in which thecondition or disorder is to be prevented.

By “subject” or “individual” or “animal” or “patient” or “mammal,” ismeant any subject, particularly a mammalian subject, for whom diagnosis,prognosis, or therapy is desired. Mammalian subjects include humans,domestic animals, farm animals, and zoo, sports, or pet animals such asdogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows,bears, and so on.

As used herein, phrases such as “a subject that would benefit fromadministration of an anti-SEMA4D antibody” and “an animal in need oftreatment” includes subjects, such as mammalian subjects, that wouldbenefit from administration of an anti-SEMA4D antibody or other SEMA4Dbinding molecule used, e.g., for detection of a SEMA4D polypeptide(e.g., for a diagnostic procedure) and/or from treatment, i.e.,palliation or prevention of a disease, with an anti-SEMA4D antibody orother SEMA4D binding molecule.

A “binding molecule” or “antigen binding molecule” of the presentinvention refers in its broadest sense to a molecule that specificallybinds an antigenic determinant. In one embodiment, the binding moleculespecifically binds to SEMA4D, e.g., to a transmembrane SEMA4Dpolypeptide of about 150 kDa or a soluble SEMA4D polypeptide of about120 kDa (commonly referred to as sSEMA4D). In another embodiment, abinding molecule of the invention is an antibody or an antigen bindingfragment thereof. In another embodiment, a binding molecule of theinvention comprises at least one heavy or light chain CDR of an antibodymolecule. In another embodiment, a binding molecule of the inventioncomprises at least two CDRs from one or more antibody molecules. Inanother embodiment, a binding molecule of the invention comprises atleast three CDRs from one or more antibody molecules. In anotherembodiment, a binding molecule of the invention comprises at least fourCDRs from one or more antibody molecules. In another embodiment, abinding molecule of the invention comprises at least five CDRs from oneor more antibody molecules. In another embodiment, a binding molecule ofthe invention comprises at least six CDRs from one or more antibodymolecules.

The present application is directed to a method of decreasing bloodbrain barrier permeability in a subject having a neuroinflammatorydisorder (e.g., Multiple Sclerosis, Amyotrophic Lateral Sclerosis,epilepsy, Alzheimer's Disease, Parkinson's Disease, meningitis, brainedema, brain trauma, and stroke), comprising administering to thesubject an anti-SEMA4D binding molecule, an anti-PlexinB1 bindingmolecule, or combination thereof.

As used herein, “anti-SEMA4D binding molecule” or “anti-PlexinB1 bindingmolecule” refers to an antibody, or antigen-binding fragment, variant,or derivative thereof. Unless specifically referring to full-sizedantibodies such as naturally occurring antibodies, the term “anti-SEMA4Dantibody” or “anti-PlexinB1 antibody” encompasses full-sized antibodiesas well as antigen-binding fragments, variants, analogs, or derivativesof such antibodies, e.g., naturally occurring antibody or immunoglobulinmolecules or engineered antibody molecules or fragments that bindantigen in a manner similar to antibody molecules.

As used herein, “inhibitor of SEMA4D interaction with a SEMA4D receptor”refers to an “anti-SEMA4D binding molecule”, an “anti-PlexinB1 bindingmolecule” as well as a small molecule inhibitor of SEMA4D or a SEMA4Dreceptor.

As used herein, “human” or “fully human” antibodies include antibodieshaving the amino acid sequence of a human immunoglobulin and includeantibodies isolated from human immunoglobulin libraries or from animalstransgenic for one or more human immunoglobulins and that do not expressendogenous immunoglobulins, as described infra and, for example, in U.S.Pat. No. 5,939,598 by Kucherlapati et al. “Human” or “fully human”antibodies also include antibodies comprising at least the variabledomain of a heavy chain, or at least the variable domains of a heavychain and a light chain, where the variable domain(s) have the aminoacid sequence of human immunoglobulin variable domain(s).

“Human” or “fully human” antibodies also include “human” or “fullyhuman” antibodies, as described above, that comprise, consistessentially of, or consist of, variants (including derivatives) ofantibody molecules (e.g., the VH regions and/or VL regions) describedherein, which antibodies or fragments thereof immunospecifically bind toa SEMA4D polypeptide or fragment or variant thereof. Standard techniquesknown to those of skill in the art can be used to introduce mutations inthe nucleotide sequence encoding a human anti-SEMA4D antibody,including, but not limited to, site-directed mutagenesis andPCR-mediated mutagenesis which result in amino acid substitutions.Preferably, the variants (including derivatives) encode less than 50amino acid substitutions, less than 40 amino acid substitutions, lessthan 30 amino acid substitutions, less than 25 amino acid substitutions,less than 20 amino acid substitutions, less than 15 amino acidsubstitutions, less than 10 amino acid substitutions, less than 5 aminoacid substitutions, less than 4 amino acid substitutions, less than 3amino acid substitutions, or less than 2 amino acid substitutionsrelative to the reference VH region, VHCDR1, VHCDR2, VHCDR3, VL region,VLCDR1, VLCDR2, or VLCDR3.

In certain embodiments, the amino acid substitutions are conservativeamino acid substitution, discussed further below. Alternatively,mutations can be introduced randomly along all or part of the codingsequence, such as by saturation mutagenesis, and the resultant mutantscan be screened for biological activity to identify mutants that retainactivity (e.g., the ability to bind a SEMA4D polypeptide, e.g., human,murine, or both human and murine SEMA4D). Such variants (or derivativesthereof) of “human” or “fully human” antibodies can also be referred toas human or fully human antibodies that are “optimized” or “optimizedfor antigen binding” and include antibodies that have improved affinityto antigen.

The terms “antibody” and “immunoglobulin” are used interchangeablyherein. An antibody or immunoglobulin comprises at least the variabledomain of a heavy chain, and normally comprises at least the variabledomains of a heavy chain and a light chain. Basic immunoglobulinstructures in vertebrate systems are relatively well understood. See,e.g., Harlow et al. (1988) Antibodies: A Laboratory Manual (2nd ed.;Cold Spring Harbor Laboratory Press).

As used herein, the term “immunoglobulin” comprises various broadclasses of polypeptides that can be distinguished biochemically. Thoseskilled in the art will appreciate that heavy chains are classified asgamma, mu, alpha, delta, or epsilon, (γ, μ, α, δ, ε) with somesubclasses among them (e.g., γ1-γ4). It is the nature of this chain thatdetermines the “class” of the antibody as IgG, IgM, IgA IgG, or IgE,respectively. The immunoglobulin subclasses (isotypes) e.g., IgG1, IgG2,IgG3, IgG4, IgA1, etc. are well characterized and are known to conferfunctional specialization. Modified versions of each of these classesand isotypes are readily discernable to the skilled artisan in view ofthe instant disclosure and, accordingly, are within the scope of theinstant invention. All immunoglobulin classes are clearly within thescope of the present invention, the following discussion will generallybe directed to the IgG class of immunoglobulin molecules. With regard toIgG, a standard immunoglobulin molecule comprises two identical lightchain polypeptides of molecular weight approximately 23,000 Daltons, andtwo identical heavy chain polypeptides of molecular weight53,000-70,000. The four chains are typically joined by disulfide bondsin a “Y” configuration wherein the light chains bracket the heavy chainsstarting at the mouth of the “Y” and continuing through the variableregion.

Light chains are classified as either kappa or lambda (κ, λ). Each heavychain class may be bound with either a kappa or lambda light chain. Ingeneral, the light and heavy chains are covalently bonded to each other,and the “tail” portions of the two heavy chains are bonded to each otherby covalent disulfide linkages or non-covalent linkages when theimmunoglobulins are generated either by hybridomas, B cells orgenetically engineered host cells. In the heavy chain, the amino acidsequences run from an N-terminus at the forked ends of the Yconfiguration to the C-terminus at the bottom of each chain.

Both the light and heavy chains are divided into regions of structuraland functional homology. The terms “constant” and “variable” are usedfunctionally. In this regard, it will be appreciated that the variabledomains of both the light (VL or VK) and heavy (VH) chain portionsdetermine antigen recognition and specificity. Conversely, the constantdomains of the light chain (CL) and the heavy chain (CH1, CH2 or CH3)confer important biological properties such as secretion, transplacentalmobility, Fc receptor binding, complement binding, and the like. Byconvention the numbering of the constant region domains increases asthey become more distal from the antigen binding site or amino-terminusof the antibody. The N-terminal portion is a variable region and at theC-terminal portion is a constant region; the CH3 and CL domains actuallycomprise the carboxy-terminus of the heavy and light chain,respectively.

As indicated above, the variable region allows the antibody toselectively recognize and specifically bind epitopes on antigens. Thatis, the VL domain and VH domain, or subset of the complementaritydetermining regions (CDRs) within these variable domains, of an antibodycombine to Rain the variable region that defines a three dimensionalantigen binding site. This quaternary antibody structure forms theantigen binding site present at the end of each arm of the Y. Morespecifically, the antigen binding site is defined by three CDRs on eachof the VH and VL chains. In some instances, e.g., certain immunoglobulinmolecules derived from camelid species or engineered based on camelidimmunoglobulins, a complete immunoglobulin molecule may consist of heavychains only, with no light chains. See, e.g., Hamers-Casterman et al.,Nature 363:446-448 (1993).

In naturally occurring antibodies, the six “complementarity determiningregions” or “CDRs” present in each antigen binding domain are short,non-contiguous sequences of amino acids that are specifically positionedto form the antigen binding domain as the antibody assumes its threedimensional configuration in an aqueous environment. The remainder ofthe amino acids in the antigen binding domains, referred to as“framework” regions, show less inter-molecular variability. Theframework regions largely adopt a β-sheet conformation and the CDRs formloops that connect, and in some cases form part of, the β-sheetstructure. Thus, framework regions act to form a scaffold that providesfor positioning the CDRs in correct orientation by inter-chain,non-covalent interactions. The antigen binding domain formed by thepositioned CDRs defines a surface complementary to the epitope on theimmunoreactive antigen. This complementary surface promotes thenon-covalent binding of the antibody to its cognate epitope. The aminoacids comprising the CDRs and the framework regions, respectively, canbe readily identified for any given heavy or light chain variable domainby one of ordinary skill in the art, since they have been preciselydefined (see below).

In the case where there are two or more definitions of a term that isused and/or accepted within the art, the definition of the term as usedherein is intended to include all such meanings unless explicitly statedto the contrary. A specific example is the use of the term“complementarity determining region” (“CDR”) to describe thenon-contiguous antigen combining sites found within the variable regionof both heavy and light chain polypeptides. This particular region hasbeen described by Kabat et al. (1983) U.S. Dept. of Health and HumanServices, “Sequences of Proteins of Immunological Interest” and byChothia and Lesk, J. Mol. Biol. 196:901-917 (1987), which areincorporated herein by reference, where the definitions includeoverlapping or subsets of amino acid residues when compared against eachother. Nevertheless, application of either definition to refer to a CDRof an antibody or variants thereof is intended to be within the scope ofthe term as defined and used herein. The appropriate amino acid residuesthat encompass the CDRs as defined by each of the above cited referencesare set forth below in Table 1 as a comparison. The exact residuenumbers that encompass a particular CDR will vary depending on thesequence and size of the CDR. Those skilled in the art can routinelydetermine which residues comprise a particular CDR given the variableregion amino acid sequence of the antibody.

TABLE 1 CDR Definitions¹ Kabat Chothia VH CDR1 31-35 26-32 VH CDR2 50-6552-58 VH CDR3  95-102  95-102 VL CDR1 24-34 26-32 VL CDR2 50-56 50-52 VLCDR3 89-97 91-96 ¹Numbering of all CDR definitions in Table 1 isaccording to the numbering conventions set forth by Kabat et al. (seebelow)

Kabat et al. also defined a numbering system for variable domainsequences that is applicable to any antibody. One of ordinary skill inthe art can unambiguously assign this system of “Kabat numbering” to anyvariable domain sequence, without reliance on any experimental databeyond the sequence itself. As used herein, “Kabat numbering” refers tothe numbering system set forth by Kabat et al. (1983) U.S. Dept. ofHealth and Human Services, “Sequence of Proteins of ImmunologicalInterest.” Unless otherwise specified, references to the numbering ofspecific amino acid residue positions in an anti-SEMA4D antibody orantigen-binding fragment, variant, or derivative thereof of the presentinvention are according to the Kabat numbering system.

Antibodies or antigen-binding fragments, variants, or derivativesthereof of the invention include, but are not limited to, polyclonal,monoclonal, multispecific and bispecific in which at least one arm isspecific for SEMA4D, human, humanized, primatized, or chimericantibodies, single-chain antibodies, epitope-binding fragments, e.g.,Fab, Fab′ and F(ab′)₂, Fd, Fvs, single-chain Fvs (scFv),disulfide-linked Fvs (sdFv), fragments comprising either a VL or VHdomain, fragments produced by a Fab expression library, andanti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodiesto anti-SEMA4D antibodies disclosed herein). ScFv molecules are known inthe art and are described, e.g., in U.S. Pat. No. 5,892,019.Immunoglobulin or antibody molecules of the invention can be of any type(e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3,IgG4, IgA1, and IgA2, etc.), or subclass of immunoglobulin molecule.

As used herein, the term “heavy chair portion” includes amino acidsequences derived from an immunoglobulin heavy chain. In certainembodiments, a polypeptide comprising a heavy chain portion comprises atleast one of: a VH domain, a CH1 domain, a hinge (e.g., upper, middle,and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or avariant or fragment thereof. For example, a binding polypeptide for usein the invention may comprise a polypeptide chain comprising a CH1domain; a polypeptide chain comprising a CH1 domain, at least a portionof a hinge domain, and a CH2 domain; a polypeptide chain comprising aCH1 domain and a CH3 domain; a polypeptide chain comprising a CH1domain, at least a portion of a hinge domain, and a CH3 domain, or apolypeptide chain comprising a CH1 domain, at least a portion of a hingedomain, a CH2 domain, and a CH3 domain. In another embodiment, apolypeptide of the invention comprises a polypeptide chain comprising aCH3 domain. Further, a binding polypeptide for use in the invention maylack at least a portion of a CH2 domain (e.g., all or part of a CH2domain). As set forth above, it will be understood by one of ordinaryskill in the art that these domains (e.g., the heavy chain portions) maybe modified such that they vary in amino acid sequence from thenaturally occurring immunoglobulin molecule.

In certain anti-SEMA4D antibodies, or antigen-binding fragments,variants, or derivatives thereof disclosed herein, the heavy chainportions of one polypeptide chain of a multimer are identical to thoseon a second polypeptide chain of the multimer. Alternatively, heavychain portion-containing monomers of the invention are not identical.For example, each monomer may comprise a different target binding site,forming, for example, a bispecific antibody.

The heavy chain portions of a binding molecule for use in the methodsdisclosed herein may be derived from different immunoglobulin molecules.For example, a heavy chain portion of a polypeptide can comprise aC_(H1) domain derived from an IgG1 molecule and a hinge region derivedfrom an IgG3 molecule. In another example, a heavy chain portion cancomprise a hinge region derived, in part, from an IgG1 molecule and, inpart, from an IgG3 molecule. In another example, a heavy chain portioncan comprise a chimeric hinge derived, in part, from an IgG1 moleculeand, in part, from an IgG4 molecule.

As used herein, the term “light chain portion” includes amino acidsequences derived from an immunoglobulin light chain, e.g., a kappa orlambda light chain. Preferably, the light chain portion comprises atleast one of a VL or CL domain.

Anti-SEMA4D antibodies, or antigen-binding fragments, variants, orderivatives thereof disclosed herein may be described or specified interms of the epitope(s) or portion(s) of an antigen, e.g., a targetpolypeptide disclosed herein (e.g., SEMA4D) that they recognize orspecifically bind. The portion of a target polypeptide that specificallyinteracts with the antigen binding domain of an antibody is an“epitope,” or an “antigenic determinant.” A target polypeptide cancomprise a single epitope, but typically comprises at least twoepitopes, and can include any number of epitopes, depending on the size,conformation, and type of antigen. Furthermore, it should be noted thatan “epitope” on a target polypeptide may be or may includenon-polypeptide elements, e.g., an epitope may include a carbohydrateside chain.

The minimum size of a peptide or polypeptide epitope for an antibody isthought to be about four to five amino acids. Peptide or polypeptideepitopes preferably contain at least seven, more preferably at leastnine and most preferably between at least about 15 to about 30 aminoacids. Since a CDR can recognize an antigenic peptide or polypeptide inits tertiary foam, the amino acids comprising an epitope need not becontiguous, and in some cases, may not even be on the same peptidechain. A peptide or polypeptide epitope recognized by anti-SEMA4Dantibodies of the present invention may contain a sequence of at least4, at least 5, at least 6, at least 7, more preferably at least 8, atleast 9, at least 10, at least 15, at least 20, at least 25, or betweenabout 15 to about 30 contiguous or non-contiguous amino acids of SEMA4D.

By “specifically binds,” it is generally meant that an antibody binds toan epitope via its antigen binding domain, and that the binding entailssome complementarity between the antigen binding domain and the epitope.According to this definition, an antibody is said to “specifically bind”to an epitope when it binds to that epitope, via its antigen bindingdomain more readily than it would bind to a random, unrelated epitope.The term “specificity” is used herein to qualify the relative affinityby which a certain antibody binds to a certain epitope. For example,antibody “A” may be deemed to have a higher specificity for a givenepitope than antibody “B,” or antibody “A” may be said to bind toepitope “C” with a higher specificity than it has for related epitope“D.”

By “preferentially binds,” it is meant that the antibody specificallybinds to an epitope more readily than it would bind to a related,similar, homologous, or analogous epitope. Thus, an antibody that“preferentially binds” to a given epitope would more likely bind to thatepitope than to a related epitope, even though such an antibody maycross-react with the related epitope.

By way of non-limiting example, an antibody may be considered to bind afirst epitope preferentially if it binds said first epitope with adissociation constant (K_(D)) that is less than the antibody's K_(D) forthe second epitope. In another non-limiting example, an antibody may beconsidered to bind a first antigen preferentially if it binds the firstepitope with an affinity that is at least one order of magnitude lessthan the antibody's K_(D) for the second epitope. In anothernon-limiting example, an antibody may be considered to bind a firstepitope preferentially if it binds the first epitope with an affinitythat is at least two orders of magnitude less than the antibody's K_(D)for the second epitope.

In another non-limiting example, an antibody may be considered to bind afirst epitope preferentially if it binds the first epitope with an offrate (k(off)) that is less than the antibody's k(off) for the secondepitope. In another non-limiting example, an antibody may be consideredto bind a first epitope preferentially if it binds the first epitopewith an affinity that is at least one order of magnitude less than theantibody's k(off) for the second epitope. In another non-limitingexample, an antibody may be considered to bind a first epitopepreferentially if it binds the first epitope with an affinity that is atleast two orders of magnitude less than the antibody's k(off) for thesecond epitope. An antibody or antigen-binding fragment, variant, orderivative disclosed herein may be said to bind a target polypeptidedisclosed herein (e.g., SEMA4D, e.g., human, murine, or both human andmurine SEMA4D) or a fragment or variant thereof with an off rate(k(off)) of less than or equal to 5×10⁻² sec⁻¹, 10⁻² sec⁻¹, 5×10⁻³ sec⁻¹or 10⁻³ sec⁻¹. More preferably, an antibody of the invention may be saidto bind a target polypeptide disclosed herein (e.g., SEMA4D, e.g.,human, murine, or both human and murine SEMA4D) or a fragment or variantthereof with an off rate (k(off)) less than or equal to 5×10⁻⁴ sec⁻¹,10⁻⁴ sec⁻¹, 5×10⁻⁵ sec⁻¹, or 10⁻⁵ sec⁻¹, 5×10⁻⁶ sec⁻¹, 10⁻⁶ sec⁻¹,5×10⁻⁶ sec⁻¹ or 10⁻⁷ sec⁻¹.

An antibody or antigen-binding fragment, variant, or derivativedisclosed herein may be said to bind a target polypeptide disclosedherein (e.g., SEMA4D, e.g., human, murine, or both human and murineSEMA4D) or a fragment or variant thereof with an on rate (k(on)) ofgreater than or equal to 10³ M⁻¹ sec⁻¹, 5×10³ M⁻¹ sec⁻¹, 10⁴ M⁻¹ sec⁻¹or 5×10⁴ M⁻¹ sec⁻¹. More preferably, an antibody of the invention may besaid to bind a target polypeptide disclosed herein (e.g., SEMA4D, e.g.,human, murine, or both human and murine SEMA4D) or a fragment or variantthereof with an on rate (k(on)) greater than or equal to 10⁵ M⁻¹ sec⁻¹,5×10⁵ M⁻¹ sec⁻¹, 10⁶ M⁻¹ sec⁻¹, or 5×10⁶ M⁻¹ sec⁻¹ or 10⁷ M⁻¹ sec⁻¹.

An antibody is said to competitively inhibit binding of a referenceantibody to a given epitope if it preferentially binds to that epitopeto the extent that it blocks, to some degree, binding of the referenceantibody to the epitope. Competitive inhibition may be determined by anymethod known in the art, for example, competition ELISA assays. Anantibody may be said to competitively inhibit binding of the referenceantibody to a given epitope by at least 90%, at least 80%, at least 70%at least 60%, or at least 50%.

As used herein, the term “affinity” refers to a measure of the strengthof the binding of an individual epitope with the CDR of animmunoglobulin molecule. See, e.g., Harlow et al. (1988) Antibodies: ALaboratory Manual (Cold Spring Harbor Laboratory Press, 2nd ed.) pages27-28. As used herein, the term “avidity” refers to the overallstability of the complex between a population of immunoglobulins and anantigen, that is, the functional combining strength of an immunoglobulinmixture with the antigen. See, e.g., Harlow at pages 29-34. Avidity isrelated to both the affinity of individual immunoglobulin molecules inthe population with specific epitopes, and also the valencies of theimmunoglobulins and the antigen. For example, the interaction between abivalent monoclonal antibody and an antigen with a highly repeatingepitope structure, such as a polymer, would be one of high avidity.

Anti-SEMA4D antibodies or antigen-binding fragments, variants, orderivatives thereof of the invention may also be described or specifiedin terms of their cross-reactivity. As used herein, the term“cross-reactivity” refers to the ability of an antibody, specific forone antigen, to react with a second antigen; a measure of relatednessbetween two different antigenic substances. Thus, an antibody is crossreactive if it binds to an epitope other than the one that induced itsformation. The cross reactive epitope generally contains many of thesame complementary structural features as the inducing epitope, and insome cases, may actually fit better than the original.

For example, certain antibodies have some degree of cross-reactivity, inthat they bind related, but non-identical epitopes, e.g., epitopes withat least 95%, at least 90%, at least 85%, at least 80%, at least 75%, atleast 70%, at least 65%, at least 60%, at least 55%, and at least 50%identity (as calculated using methods known in the art and describedherein) to a reference epitope. An antibody may be said to have littleor no cross-reactivity if it does not bind epitopes with less than 95%,less than 90%, less than 85%, less than 80%, less than 75%, less than70%, less than 65%, less than 60%, less than 55%, and less than 50%identity (as calculated using methods known in the art and describedherein) to a reference epitope. An antibody may be deemed “highlyspecific” for a certain epitope, if it does not bind any other analog,ortholog, or homolog of that epitope.

Anti-SEMA4D binding molecules, e.g., antibodies or antigen-bindingfragments, variants or derivatives thereof, of the invention may also bedescribed or specified in terms of their binding affinity to apolypeptide of the invention, e.g., SEMA4D, e.g., human, murine, or bothhuman and murine SEMA4D. Preferred binding affinities include those witha dissociation constant or Kd less than 5×10⁻² M, 10⁻² M, 5×10⁻³ M, 10⁻³M, S×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷ M, 10⁻⁷M, 5×10 ⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M,5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M,or 10⁻¹⁵ M. In certain embodiments, the anti-SEMA4D binding molecule,e.g., an antibody or antigen binding fragment thereof, of the inventionbinds human SEMA4D with a Kd of about 5×10⁻⁹ to about 6×10⁻⁹. In anotherembodiment, the anti-SEMA41D binding molecule, e.g., an antibody orantigen binding fragment thereof, of the invention binds murine SEMA4Dwith a Kd of about 1×10⁻⁹ to about 2×10⁻⁹.

As used herein, the term “chimeric antibody” will be held to mean anyantibody wherein the immunoreactive region or site is obtained orderived from a first species and the constant region (which may beintact, partial or modified in accordance with the instant invention) isobtained from a second species. In preferred embodiments the targetbinding region or site will be from a non-human source (e.g., mouse orprimate) and the constant region is human.

As used herein, the term “engineered antibody” refers to an antibody inwhich the variable domain in either the heavy or light chain or both isaltered by at least partial replacement of one or more CDRs from anantibody of known specificity and, if necessary, by partial frameworkregion replacement and sequence changing. Although the CDRs may bederived from an antibody of the same class or even subclass as theantibody from which the framework regions are derived, it is envisagedthat the CDRs will be derived from an antibody of different class andpreferably from an antibody from a different species. An engineeredantibody in which one or more “donor” CDRs from a non-human antibody ofknown specificity is grafted into a human heavy or light chain frameworkregion is referred to herein as a “humanized antibody.” It may not benecessary to replace all of the CDRs with the complete CDRs from thedonor variable domain to transfer the antigen binding capacity of onevariable domain to another. Rather, it may only be necessary to transferthose residues that are necessary to maintain the activity of the targetbinding site.

It is further recognized that the framework regions within the variabledomain in a heavy or light chain, or both, of a humanized antibody maycomprise solely residues of human origin, in which case these frameworkregions of the humanized antibody are referred to as “fully humanframework regions” (for example, MAb VX15/2503, disclosed in U.S. PatentAppl. Publication No. US 2010/0285036 A1 as MAb 2503, incorporatedherein by reference in its entirety). Alternatively, one or moreresidues of the framework region(s) of the donor variable domain can beengineered within the corresponding position of the human frameworkregion(s) of a variable domain in a heavy or light chain, or both, of ahumanized antibody if necessary to maintain proper binding or to enhancebinding to the SEMA4D antigen. A human framework region that has beenengineered in this manner would thus comprise a mixture of human anddonor framework residues, and is referred to herein as a “partiallyhuman framework region.”

For example, humanization of an anti-SEMA4D antibody can be essentiallyperformed following the method of Winter and co-workers (Jones et al.,Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988);Verhoeyen et al., Science 239:1534-1536 (1988)), by substituting rodentor mutant rodent CDRs or CDR sequences for the corresponding sequencesof a human anti-SEMA4D antibody. See also U.S. Pat. Nos. 5,225,539;5,585,089; 5,693,761; 5,693,762; 5,859,205; herein incorporated byreference. The resulting humanized anti-SEMA4D antibody would compriseat least one rodent or mutant rodent CDR within the fully humanframework regions of the variable domain of the heavy and/or light chainof the humanized antibody. In some instances, residues within theframework regions of one or more variable domains of the humanizedanti-SEMA4D antibody are replaced by corresponding non-human (forexample, rodent) residues (see, for example, U.S. Pat. Nos. 5,585,089;5,693,761; 5,693,762; and 6,180,370), in which case the resultinghumanized anti-SEMA4D antibody would comprise partially human frameworkregions within the variable domain of the heavy and/or light chain.

Furthermore, humanized antibodies can comprise residues that are notfound in the recipient antibody or in the donor antibody. Thesemodifications are made to further refine antibody performance (e.g., toobtain desired affinity). In general, the humanized antibody willcomprise substantially all of at least one, and typically two, variabledomains, in which all or substantially all of the CDRs correspond tothose of a non-human immunoglobulin and all or substantially all of theframework regions are those of a human immunoglobulin sequence. Thehumanized antibody optionally also will comprise at least a portion ofan immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details see Jones et al., Nature 331:522-525(1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr.Op. Struct. Biol. 2:593-596 (1992); herein incorporated by reference.Accordingly, such “humanized” antibodies may include antibodies whereinsubstantially less than an intact human variable domain has beensubstituted by the corresponding sequence from a non-human species. Inpractice, humanized antibodies are typically human antibodies in whichsome CDR residues and possibly some framework residues are substitutedby residues from analogous sites in rodent antibodies. See, for example,U.S. Pat. Nos. 5,225,539; 5,585,089; 5,693,761; 5,693,762; 5,859,205.See also U.S. Pat. No. 6,180,370, and International Publication No. WO01/27160, where humanized antibodies and techniques for producinghumanized antibodies having improved affinity for a predeterminedantigen are disclosed.

II. Blood Brain Barrier (“BBB”)

The blood-brain barrier (BBB) is an active interface between circulatingblood and the central nervous system (CNS). The BBB restricts the freemovement of different substances between the two compartments and playsa crucial role in the maintenance of the homeostasis of the CNS. The BBBhas both a barrier function and a carrier function. As a barrier, theBBB restricts the movement of cells and potentially toxic or harmfulsubstances from the blood to the brain. As a carrier, on the other hand,the BBB is responsible for transporting nutrients to the brain andremoving metabolites.

The BBB is primarily composed of three components: endothelial cells,astrocytes, and pericytes. Endothelial cells form a continuous sheetcovering the inner surface of the capillaries and blood vessels in thebrain. (Ransohoff et al., “Three or More Routes for Leukocyte MigrationInto the Central Nervous System,” Nature Rev. Immun. 3:569-581 (2003).The endothelial cells are located adjacent to the basal membrane, whichconsists mainly of collagen IV, fibronectin, laminin and proteoglycans,and are interconnected by tight junctions that form a belt-likestructure at the apical region of the cells. Endothelial cells restrictthe diffusion of microscopic objects (e.g. bacteria) and large orhydrophilic molecules into the brain parenchyma and cerebrospinal fluid(CSF), while allowing the diffusion of small hydrophobic molecules (O₂,hormones, CO₂). Cells of the barrier actively transport metabolicproducts such as glucose across the barrier with specific proteins.

The endothelial cells which form the brain capillaries are differentfrom those found in other tissues in the body. Brain capillaryendothelial cells are joined together by tight intercellular junctionsthat form a continuous wall against the passive diffusion of moleculesfrom the blood to the brain and other parts of the CNS (includingCerebrospinal Fluid, CSF). These cells are also different in that theyhave few pinocytic vesicles which in other tissues allow somewhatunselective transport across the capillary wall. Also lacking arecontinuous gaps or channels running between the cells which would allowunrestricted passage.

In addition to endothelial cells, the BBB is also composed of pericytesand astrocytes. Pericytes are located within the basal membrane,interact with endothelial cells and play an important role in theregulation of endothelial proliferation, angiogenesis and inflammatoryprocesses. Astrocytes are characteristic star-shaped glial cells in thebrain and spinal cord and are the most abundant cell of the human brain.They perform many functions, including biochemical support ofendothelial cells that form the blood-brain barrier, provision ofnutrients to the nervous tissue, maintenance of extracellular ionbalance, and a role in the repair and scarring process of the brain andspinal cord following traumatic injuries.

The blood-brain barrier functions to ensure that the environment of thebrain is constantly controlled. The levels of various substances in theblood, such as hormones, amino acids, and ions, undergo frequent smallfluctuations which can be brought about by activities such as eating andexercise (Goldstein et al., “The Blood-Brain Barrier,” ScientificAmerican 255:74-83 (1986); Pardridge, “Receptor-Mediated PeptideTransport Through the Blood-Brain Barrier,” Endocrin. Rev. 7:314-330(1986)). If the brain was not protected by the blood brain barrier fromthese variations in serum composition, the result could be uncontrolledneural activity.

The isolation of the brain from the bloodstream is not complete. If thiswere the case, the brain would be unable to function properly due to alack of nutrients and because of the need to exchange chemicals with therest of the body. The presence of specific transport systems within thecapillary endothelial cells assures that the brain receives, in acontrolled manner, all of the compounds required for normal growth andfunction. In many instances, these transport systems consist ofmembrane-associated proteins, which selectively bind and transportcertain molecules across the barrier membranes. These transporterproteins are known as solute carrier transporters.

Although the BBB serves to protect the brain and the central nervoussystem from damage from foreign or external molecules and cells, foreignor external molecules and cells can often cross the BBB and, in limitednumbers, may even be beneficial such as for immune surveillance of theCNS. However, when highly active cells, such as, for instance, B cells,T cells, leukocytes and macrophages, cross the BBB in excess and reachthe brain, they can cause damage to the brain. Patients suffering fromedema, brain traumas, stroke and multiple sclerosis, for instance,exhibit a breakdown of the BBB.

The effect of the BBB on various neuroinflammatory disorders has beenstudied. (Zlokovic B V, “The Blood-Brain Barrier in Health and ChronicNeurodegenerative Disorders,” Neuron 57: 178-201 (2008); Zhong Z et al.,“ALS-causing SOD1 mutants generate vascular changes prior to motorneuron degeneration,” Nature Neuroscience 11(4): 420-422 (2008); HawkinsB T et al., “The Blood-Brain Barrier/neurovascular Unit in Health andDisease,” Pharmacological Rev 57 (2): 173-185 (2005); Oby E et al., “TheBlood-Brain Barrier and Epilepsy,” Epilepsia 47(11); 1761-1774 (2006)).Moreover, there is increasing evidence that inflammation and theblood-brain barrier (BBB) (Banks and Erickson, 2010; Lochhead et al,2010) are involved in the pathogenesis of neurologic diseases such asmeningitis (van der et al, 2004), brain edema (Stamatovic et al, 2006),Alzheimer's disease (Kalaria, 1992), Parkinson's disease (Westin, J. E.,et. al., “Endothelial Proliferation and Increased Blood-Brain BarrierPermeability it the Basal Ganglia in a Rat Model of3,4-Dihydroxyphenyl-L-Alanine-Induced Dyskinesia,” The Journal ofNeuroscience 26(37): 9448-9461 (2006)) and multiple sclerosis (Minagarand Alexander, 2003).

In the case of multiple sclerosis, for instance, it has been shown usingMagnetic Resonance Imaging (“MRI”), that when a person is undergoing anMS “attack,” the BBB has broken down in a section of the brain or spinalcord, allowing T lymphocytes to cross over and attack the myelin thatprotects and insulates the neurons of the central nervous systems inboth brain and spinal cord. (Zlokovic 2008; Waubant E., “Biomarkersindicative of blood-brain barrier disruption in multiple sclerosis”.Disease Markers 22 (4): 235-44 (2006)).

Meningitis, on the other hand, occurs when there is an inflammation ofthe membranes that surround the brain and spinal cord (these membranesare known as meninges). When the meninges are inflamed, the blood-brainbarrier may be disrupted, allowing both inflammatory cells and varioussubstances (including either toxins or antibiotics) to enter the brain.(Beam, T R Jr., et al. (December 1977). “Blood, brain, and cerebrospinalfluid concentrations of several antibiotics in rabbits with intact andinflamed meninges”. Antimicrobial Agents and Chemotherapy 12 (6):710-6).

Similarly, in the case of Parkinson's Disease (PD), it has beensuggested that absorption or metabolism of putative PD toxins, and theirfaulty elimination across the BBB, due to low activity of thetransporter P-glycoportein (P-gp), an ATP-dependent efflux pump whichmediates rapid removal of ingested toxic lipophilic metabolites, mayplay a role in the pathogenesis of PD (Kortekaas, R., Leenders, K. L.,van Oostrom, J. C., Vaalburg, W., Bart, J., Willemsen, A. T., andHendrikse, N. H. Blood-brain barrier dysfunction in parkinsonianmidbrain in vivo. Ann. Neurol. 57, 176-179, 2005). Neuroinflammationalso appears to be a ubiquitous finding in PD patients and experimentalmodels of PD. Phagocyte activation, increased synthesis and release ofproinflammatory cytokines, complement activation, activation ofmicroglia, and release of reactive oxygen species (ROS) have beendescribed (Whitton, P. S. Inflammation as a causative factor in theaetiology of Parkinson's disease. Br. J. Pharmacol. 150, 963-976, 2007).

In epilepsy, studies have implicated the failure of blood-brain barrierfunction in triggering chronic or acute seizures due to certaininteractions between a common blood protein, albumin, and astrocytes.These findings suggest that acute seizures are a result of disruption ofthe BBB by either artificial or inflammatory mechanisms. (Oby, E; et al.(2006). “The Blood-Brain Barrier and Epilepsy” (PDF). Epilepsia 47 (11):1761-1774).

In patients with Alzheimer's Disease (AD), evidence points to thedisruption of the blood-brain barrier in allowing blood plasmacontaining amyloid beta (Aβ) to enter the brain though RAGE, a majorinflux transporter for Aβ across the BBB. Studies have shown that theAβ/RAGE interaction results in transcytosis of circulating Aβ across theBBB into the brain parenchyma and its binding to neurons, NF-kB-mediatedendothelial activation resulting in secretion of proinflammatorycytokines, the expression of adhesion molecules, and the generation ofendothelin-1, which suppresses CBF (Cerebral Blood Flow). Moreover, ithas been shown that the Aβ/RAGE interaction contributes to neuronalkilling by producing oxidative damage to RAGE-expressing neurons and byactivating microglia. (Zlokovic, B. V. The Blood-Brain Barrier in Healthand Chronic Neurodegenerative Disorders. Neuron 57, 178-201, 2008).Faulty efflux of Aβ out of the brain parenchyma and into themicrovasculature via the BBB has also been found in the setting of ADpathogenesis and has been attributed, in part, to compromisedlow-density lipoprotein receptor related protein 1 (LRP1) function. LRP1is an abluminal BBB membrane protein that binds and transports differentstructural conformers of Aβ (Deane et al., “LRP/amyloid beta-peptideinteraction mediates differential brain efflux of Abeta isoforms.”Neuron 43, 333-344, 2004). Aβ exposure shifts cell surface expressionpatterns of tight junction proteins, including claudin-5 and ZO-2, onbrain microvascular endothelial cells to the cytoplasm (Marco et al.,“Amyloid β-peptide 1-42 alters tight junction protein distribution andexpression in brain microvessel endothelial cells.” Neurosci. Lett. 401,219-224, 2006), and severely compromises transendothelial electricalresistance (TEER) of monolayers of these cells (Gonzalez-Velasquez etal., “Soluble aggregates of the amyloid-beta protein selectivelystimulate permeability in human brain microvascular endothelialmonolayers.” J. Neurochem. 107, 466-477, 2008).

In Amyotrophic lateral sclerosis (ALS), studies have suggested that BBBbreakdown may result in leakage of serum proteins that interact withmotor neurons to produce ROS (Reactive Oxygen Species) and initiate anautoimmune response, causing demyelination, disruption of neuronaltransmission, and cell death. (Zlokovic 2008).

A recent study suggests that the weakening of the BBB can result from adisturbance in endothelial cells mediated through their VEGF-A receptor.(Argaw A T et al., “VEGF-mediated disruption of endothelial CLN-5promotes blood-brain barrier breakdown,” PNAS 106(6): 1977-1982 (2009)).According to that study, VEGF-A, which is derived from astrocytes,targets and disrupts expression of both endothelial transmembrane tightjunction proteins claudin-5 (CLN-5) and occludin (OCLN). As expressionof both CLN-5 and OCLN decreases, breakdown of the BBB increases.

As shown in the present examples, another possible mechanism for theweakening of the BBB is as a result of endothelial cell disturbancethrough the Plexin-B1 high affinity (1 nM) receptor for SEMA4D.Plexin-B1 can be expressed by endothelial cells. In the presence ofSEMA4-D, endothelial cells may undergo a transformation which alters themorphology or function of the endothelial cells so as to cause aweakening of the BBB, for example, through modification of tightjunctions. This weakening of the BBB may, in turn, increase permeabilityof the BBB to cells and molecules and allow such cells and molecules toenter and alter activity of the brain and central nervous system.Addition of either anti-SEMA4D or anti-Plexin-B1, consequently, mayprevent endothelial cells from undergoing a transformation and reduceweakening of the BBB.

III. Target Polypeptide Description

As used herein, the terms “semaphorin-4D,” “SEMA4D” and “SEMA4Dpolypeptide” are used interchangeably, as are “SEMA4D” and “Sema4D.” Incertain embodiments, SEMA4D is expressed on the surface of or secretedby a cell. In another embodiment, SEMA4D is membrane bound. In anotherembodiments, SEMA4D is soluble, e.g., sSEMA4D. In other embodiments,SEMA4D may include a full-sized SEMA4D or a fragment thereof, or aSEMA4D variant polypeptide, wherein the fragment of SEMA4D or SEMA4Dvariant polypeptide retains some or all functional properties of thefull-sized SEMA4D.

The full-sized human SEMA4D protein is a homodimeric transmembraneprotein consisting of two polypeptide chains of 150 kDa. SEMA4D belongsto the semaphorin family of cell surface receptors and is also referredto as CD100. Both human and mouse SEMA4D/Sema4D are proteolyticallycleaved from their transmembrane form to generate 120-kDa soluble forms,indicating the existence of two Sema4D isoforms (Kumanogoh et al., J.Cell Science 6(7):3464 (2003)). Semaphorins include soluble andmembrane-bound proteins that were originally defined as axonal-guidancefactors during development which play an important role in establishingprecise connections between neurons and their appropriate target.Structurally considered a class IV semaphorin, full-sized SEMA4Dincludes an amino-terminal signal sequence followed by a characteristic‘Sema’ domain, which contains 17 conserved cysteine residues, an Ig-likedomain, a lysine-rich stretch, a hydrophobic transmembrane region, and acytoplasmic tail.

Each polypeptide chain of SEMA4D includes a signal sequence of about 13amino acids followed by a semaphorin domain of about 512 amino acids, animmunoglobulin-like (Ig-like) domain of about 65 amino acids, alysine-rich stretch of 104 amino acids, a hydrophobic transmembraneregion of about 19 amino acids, and a cytoplasmic tail of 110 aminoacids. A consensus site for tyrosine phosphorylation in the cytoplasmictail supports the predicted association of SEMA4D with a tyrosine kinase(Schlossman, et al., Eds. (1995) Leucocyte Typing V (Oxford UniversityPress, Oxford)).

SEMA4D is known to have at least two receptors. One of the receptors,Plexin-B1, is expressed in non-lymphoid tissues and has been shown to bea high affinity (1 nM) receptor for SEMA4D (Tamagnone et al., Cell99:71-80 (1999)). SEMA4D stimulation of Plexin-B1 signaling has beenshown to induce growth cone collapse of neurons, and to induce processextension collapse and apoptosis of oligodendrocytes (Giraudon et al.,J. Immunol. 172:1246-1255 (2004); Giraudon et al., NeuroMolecular Med.7:207-216 (2005)). After binding to SEMA4D, Plexin-B1 signaling mediatesthe inactivation of R-Ras, leading to a decrease in the integrinmediated attachment to the extracellular matrix, as well as toactivation of RhoA, leading to reorganization of the cytoskeleton andcell migration. See Kruger et al., Nature Rev. Mol. Cell. Biol.6:789-800 (2005); Pasterkamp, TRENDS in Cell Biology 15:61-64 (2005)).

In lymphoid tissues CD72 is utilized as a low affinity (300 nM) SEMA4Dreceptor (Kumanogoh et al., Immunity 13:621-631 (2000)). B cells andAPCs express CD72, and anti-CD72 antibodies have many of the sameeffects as sSEMA4D, such as enhancement of CD40-induced B cell responsesand B cell shedding of CD23. CD72 is thought to act as a negativeregulator of B cell responses by recruiting the tyrosine phosphataseSHP-1, which can associate with many inhibitory receptors. Interactionof SEMA4D with CD72 results in the dissociation of SHP-1, and the lossof this negative activation signal. SEMA4D has been shown to promote Tcell stimulation and B cell aggregation and survival in vitro. Theaddition of SEMA4D-expressing cells or sSEMA4D enhances CD40-induced Beeli proliferation and immunoglobulin production in vitro, andaccelerates in vivo antibody responses (Ishida et al., Inter. Immunol.15:1027-1034 (2003); Kumanogoh and H. Kukutani, Trends in Immunol.22:670-676 (2001)). sSEMA4D enhances the CD40 induced maturation of DCs,including up-regulation of costimulatory molecules and increasedsecretion of IL-12. In addition, sSEMA4D can inhibit immune cellmigration, which can be reversed by addition of blocking anti-SEMA4Dantibodies (Elhabazi et al., J. Immunol. 166:4341-4347 (2001); Delaireet al., J. Immunol. 166:4348-4354 (2001)).

Sema4D is expressed at high levels in lymphoid organs, including thespleen, thymus, and lymph nodes, and in non-lymphoid organs, such as thebrain, heart, and kidney. In lymphoid organs, Sema4D is abundantlyexpressed on resting T cells but only weakly expressed on resting Bcells and antigen-presenting cells (APCs), such as dendritic cells(DCs). Cellular activation increases the surface expression of SEMA4D aswell as the generation of soluble SEMA4D (sSEMA4D).

The expression pattern of SEMA4D suggests that it plays an importantphysiological role as well as pathological role in the immune system.SEMA4D has been shown to promote B cell activation, aggregation andsurvival; enhance CD40-induced proliferation and antibody production;enhance antibody response to T cell dependent antigens; increase T cellproliferation; enhance dendritic cell maturation and ability tostimulate T cells; and is directly implicated in demyelination andaxonal degeneration (Shi et al., Immunity 13:633-642 (2000); Kumanogohet al., J Immunol 169:1175-1181 (2002); and Watanabe et al., J Immunol167:4321-4328 (2001)).

SEMA4D knock out (SEMA4D−/−) mice have provided additional evidence thatSEMA4D plays an important role in both humoral and cellular immuneresponses. There are no known major abnormalities of non-lymphoidtissues in SEMA4D−/− mice. Dendritic cells (DCs) from the SEMA4D−/− micehave poor allostimulatory ability and show defects in expression ofcostimulatory molecules, which can be rescued by the addition ofsSEMA4D. Mice deficient in SEMA4D (SEMA4D−/−) fail to developexperimental autoimmune encephalomyelitis induced by myelinoligodendrocyte glycoprotein peptide, because myelin oligodendrocyteglycoprotein-specific Tcells are poorly generated in the absence ofSEMA4D (Kumanogoh et al., J Immunol 169:1175-1181 (2002)). A significantamount of soluble SEMA4D is also detected in the sera ofautoimmunity-prone MRL/lpr mice (model of systemic autoimmune diseasessuch as SLE), but not in normal mice. Further, the levels of sSEMA4Dcorrelate with levels of auto-antibodies and increase with age (Wang etal., Blood 97:3498-3504 (2001)). Soluble SEMA4D has also been shown toaccumulate in the cerebral spinal fluid and sera of patients withdemyelinating disease, and sSEMA4D induces apoptosis of humanpluripotent neural precursors (Dev cells), and both inhibits processextension and induces apoptosis of rat oligodendrocytes in vitro(Giraudon et al., J Immunol 172(2):1246-1255 (2004)). This apoptosis wasblocked by an anti-SEMA4D MAb.

IV. Anti-SEMA4D Antibodies

Antibodies that bind SEMA4D have been described in the art. See, forexample, US Publ. Nos. 2008/0219971 A1, US 2010/0285036 A1, and US2006/0233793 A1, International Patent Applications WO 93/14125, WO2008/100995 and WO 2010/129917, and Herold et al., Int. Immunol. 7(1):1-8 (1995), each of which is herein incorporated in its entirety byreference.

The present application generally relates to a method of decreasingblood brain barrier permeability in a subject, e.g., a human patient,having a neuroinflammatory disorder, e.g., a CNS inflammatory disorderor neurodegenerative disorder, comprising administration of an antibodywhich specifically binds to SEMA4D, or an antigen-binding fragment,variant, or derivative thereof. In certain embodiments, the antibodyblocks the interaction of SEMA4D with one or more of its receptors,e.g., Plexin-B1. Anti-SEMA4D antibodies having these properties can beused in the methods provided herein. Antibodies that can be usedinclude, but are not limited to MAbs VX15/2503, 67, and 76 andantigen-binding fragments, variants, or derivatives thereof which arefully described in US 2010/0285036 A1. Additional antibodies which canbe used in the methods provided herein include the BD16 and BB18antibodies described in US 2006/0233793 A1 as well as antigen-bindingfragments, variants, or derivatives thereof; or any of MAb 301, MAb1893, MAb 657, MAb 1807, MAb 1656, MAb 1808, Mab 59, MAb 2191, MAb 2274,MAb 2275, MAb 2276, MAb 2277, MAb 2278, MAb 2279, MAb 2280, MAb 2281,MAb 2282, MAb 2283, MAb 2284, and MAb 2285, as well as any fragments,variants or derivatives thereof as described in US 2008/0219971 A1. Incertain embodiments an anti-SEMA4D antibody for use in the methodsprovided herein binds human, murine, or both human and murine SEMA4D.Also useful are antibodies which bind to the same epitope as any of theaforementioned antibodies and/or antibodies which competitively inhibitany of the aforementioned antibodies from binding to SEMA4D.

In certain embodiments, an anti-SEMA4D antibody or antigen-bindingfragment, variant, or derivative thereof useful in the methods providedherein has an amino acid sequence that has at least about 80%, about85%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%,about 94%, or about 95% sequence identity to the amino acid sequence fora reference anti-SEMA4D antibody molecule, for example those describedabove. In a further embodiment, the binding molecule shares at leastabout 96%, about 97%, about 98%, about 99%, or 100% sequence identity toa reference antibody.

In another embodiment, an anti-SEMA4D antibody or antigen-bindingfragment, variant, or derivative thereof useful in the methods providedherein comprises, consists essentially of, or consists of animmunoglobulin heavy chain variable domain (VII domain), where at leastone of the CDRs of the VH domain has an amino acid sequence that is atleast about 80%, about 85%, about 90%, about 95%, about 96%, about 97%,about 98%, about 99%, or identical to CDR1, CDR2 or CDR3 of SEQ ID NO: 9or 10.

In another embodiment, an anti-SEMA4D antibody or antigen-bindingfragment, variant, or derivative thereof useful in the methods providedherein comprises, consists essentially of, or consists of animmunoglobulin heavy chain variable domain (VH domain), where at leastone of the CDRs of the VH domain has an amino acid sequence that is atleast about 80%, about 85%, about 90%, about 95%, about 96%, about 97%,about 98%, about 99%, or identical to SEQ ID NO: 6, SEQ ID NO: 7, or SEQID NO: 8.

In another embodiment, an anti-SEMA4D antibody or antigen-bindingfragment, variant, or derivative thereof useful in the methods providedherein comprises, consists essentially of, or consists of animmunoglobulin heavy chain variable domain (VH domain), where at leastone of the CDRs of the VH domain has an amino acid sequence identical,except for 1, 2, 3, 4, or 5 conservative amino acid substitutions, toSEQ NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8.

in another embodiment, an anti-SEMA4D antibody or antigen-bindingfragment, variant, or derivative thereof useful in the methods providedherein comprises, consists essentially of or consists of a VH domainthat has an amino acid sequence that is at least about 80%, about 85%,about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about96%, about 97%, about 98%, about 99%, or 100% identical to SEQ ID NO: 9or SEQ ID NO: 10, wherein the anti-SEMA4D antibody comprising theencoded VII domain specifically, preferentially, or competitively bindsto SEMA4D.

In another embodiment, an anti-SEMA4D antibody or antigen-bindingfragment, variant, or derivative thereof useful in the methods providedherein comprises, consists essentially of, or consists of animmunoglobulin light chain variable domain (VL domain), where at leastone of the CDRs of the VL domain has an amino acid sequence that is atleast about 80%, about 85%, about 90%, about 95%, about 96%, about 97%,about 98%, about 99%, or identical to CDR1, CDR2 or CDR3 of SEQ ID NO:17 or 18.

In another embodiment, an anti-SEMA4D antibody or antigen-bindingfragment, variant, or derivative thereof useful in the methods providedherein comprises, consists essentially of, or consists of animmunoglobulin light chain variable domain (VL domain), where at leastone of the CDRs of the VL domain has an amino acid sequence that is atleast about 80%, about 85%, about 90%, about 95%, about 96%, about 97%,about 98%, about 99%, or identical to SEQ ID NO: 14, SEQ ID NO: 15, orSEQ ID NO: 16.

In another embodiment, an anti-SEMA4D antibody or antigen-bindingfragment, variant, or derivative thereof useful in the methods providedherein comprises, consists essentially of, or consists of animmunoglobulin light chain variable domain (VL domain), where at leastone of the CDRs of the VL domain has an amino acid sequence identical,except for 1, 2, 3, 4, or 5 conservative amino acid substitutions, toSEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16.

In another embodiment, an anti-SEMA4D antibody or antigen-bindingfragment, variant, or derivative thereof useful in the methods providedherein comprises, consists essentially of, or consists of a VL domainthat has an amino acid sequence that is at least about 80%, about 85%,about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about96%, about 97%, about 98%, about 99%, or 100% identical to SEQ ID NO: 17or SEQ ID NO: 18, wherein the anti-SEMA4D antibody comprising theencoded VL domain specifically, preferentially, or competitively bindsto SEMA4D.

In another embodiment, an anti-SEMA4D antibody or antigen-bindingfragment, variant, or derivative thereof useful in the methods providedherein comprises, consists essentially of, or consists of animmunoglobulin heavy chain variable domain (VH domain) and animmunoglobulin light chain variable domain (VL domain), where at leastone of the CDRs of the VH domain has an amino acid sequence that is atleast about 80%, about 85%, about 90%, about 95%, about 96%, about 97%,about 98%, about 99%, or identical to CDR1, CDR2 or CDR3 of SEQ ID NO: 9or 10 and at least one of the CDRs of the VL domain has an amino acidsequence that is at least about 80%, about 85%, about 90%, about 95%,about 96%, about 97%, about 98%, about 99%, or identical to CDR1, CDR2or CDR3 of SEQ ID NO: 17 or 18.

In another embodiment, an anti-SEMA4D antibody or antigen-bindingfragment, variant, or derivative thereof useful in the methods providedherein comprises, consists essentially of, or consists of animmunoglobulin heavy chain variable domain (VH domain) and animmunoglobulin light chain variable domain (VL domain), where at leastone of the CDRs of the VH domain has an amino acid sequence identical,except for 1, 2, 3, 4, or 5 conservative amino acid substitutions, toSEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8 and where at least one ofthe CDRs of the VL domain has an amino acid sequence identical, exceptfor 1, 2, 3, 4, or 5 conservative amino acid substitutions, to SEQ IDNO: 14, SEQ ID NO: 15, or SEQ ID NO: 16.

In another embodiment, an anti-SEMA4D antibody or antigen-bindingfragment, variant, or derivative thereof useful in the methods providedherein comprises, consists essentially of, or consists of a VH domainthat has an amino acid sequence that is at least about 80%, about 85%,about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about96%, about 97%, about 98%, about 99%, or 100% identical to SEQ ID NO: 9or SEQ ID NO: 10, and a VL domain that has an amino acid sequence thatis at least about 80%, about 85%, about 90%, about 91%, about 92%, about93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%,or 100% identical to SEQ ID NO: 17 or SEQ ID NO: 18, wherein theanti-SEMA4D antibody comprising the encoded VH and VL domainsspecifically, preferentially, or competitively hinds to SEMA4D.

In another embodiment, an anti-SEMA4D antibody or antigen-bindingfragment, variant, or derivative thereof useful in the methods providedherein comprises, consists essentially of, or consists of the three CDRsof the VL domain and three CDRs of the VH domain of MAb VX15/2503, 67,or 76, which are fully described in US 2010/0285036 A1 in someembodiments, the anti-SEMA4D antibody useful in the methods providedherein comprises MAb VX15/2503 or 67.

Also included for use in the methods provided herein are polypeptidesencoding anti-SEMA4D antibodies, or antigen-binding fragments, variants,or derivatives thereof as described herein, polynucleotides encodingsuch polypeptides, vectors comprising such polynucleotides, and hostcells comprising such vectors or polynucleotides, all for producinganti-SEMA4D antibodies, or antigen-binding fragments, variants, orderivatives thereof for use in the methods described herein.

Suitable biologically active variants of the anti-SEMA4D antibodies ofthe invention can be used in the methods of the present invention. Suchvariants will retain the desired binding properties of the parentanti-SEMA4D antibody. Methods for making antibody variants are generallyavailable in the art.

Methods for mutagenesis and nucleotide sequence alterations are wellknown in the art See, for example, Walker and Gaastra, eds. (1983)Techniques in Molecular Biology (MacMillan Publishing Company, NewYork); Kunkel, Proc. Natl. Acad. Sci. USA 82:488-492 (1985); Kunkel etal., Methods Enzymol. 154:367-382 (1987); Sambrook et al. (1989)Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, N.Y.); U.S.Pat. No. 4,873,192; and the references cited therein; hereinincorporated by reference. Guidance as to appropriate amino acidsubstitutions that do not affect biological activity of the polypeptideof interest may be found in the model of Dayhoff et al. (1978) in Atlasof Protein Sequence and Structure (Natl. Biomed. Res. Found.,Washington, D.C.), pp. 345-352, herein incorporated by reference in itsentirety. The model of Dayhoff et al. uses the Point Accepted Mutation(PAM) amino acid similarity matrix (PAM 250 matrix) to determinesuitable conservative amino acid substitutions. Conservativesubstitutions, such as exchanging one amino acid with another havingsimilar properties, may be preferred. Examples of conservative aminoacid substitutions as taught by the PAM 250 matrix of the Dayhoff et al.model include, but are not limited to, Gly

Ala, Val

Ile

Leu, Asp

Glu, Lys

Arg, Asn

Gln, and Phe

Trp

Tyr.

In constructing variants of the anti-SEMA4D binding molecule, e.g., anantibody or antigen-binding fragment thereof, polypeptides of interest,modifications are made such that variants continue to possess thedesired properties, e.g., being capable of specifically binding to aSEMA4D, e.g., human, murine, or both human and murine SEMA4D, e.g.,expressed on the surface of or secreted by a cell and having SEMA4Dblocking activity, as described herein. Obviously, any mutations made inthe DNA encoding the variant polypeptide must not place the sequence outof reading frame and preferably will not create complementary regionsthat could produce secondary mRNA structure. See EP Patent ApplicationPublication No. 75,444.

Methods for measuring anti-SEMA4D binding molecule, e.g., an antibody orantigen-binding fragment, variant, or derivative thereof, bindingspecificity include, but are not limited to, standard competitivebinding assays, assays for monitoring immunoglobulin secretion by cellsor B cells. T cell proliferation assays, apoptosis assays, ET ISAassays, and the like. See, for example, such assays disclosed in WO93/14125; Shi et al., Immunity 13:633-642 (2000); =Kumnanogoh et al., JImmunol 169:1175-1181 (2002); Watanabe et al., J Immunol 167:4321-4328(2001); Wang et al., Blood 97:3498-3504 (2001); and Giraudon et al., JImmunol 172(2):1246-1255 (2004), all of which are herein incorporated byreference.

When discussed herein whether any particular polypeptide, including theconstant regions, CDRs, VH domains, or VL domains disclosed herein, isat least about 65%, about 70%, about 75%, about 80%, about 85%, about90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,about 97%, about 98%, about 99%, or even about 100% identical to anotherpolypeptide, the % identity can be determined using methods and computerprograms/software known in the art such as, but not limited to, theBESTFIT program (Wisconsin Sequence Analysis Package, Version 8 forUnix, Genetics Computer Group, University Research Park, 575 ScienceDrive, Madison, Wis. 53711). BESTFIT uses the local homology algorithmof Smith and Waterman (1981) Adv. Appl. Math. 2:482-489, to find thebest segment of homology between two sequences. When using BESTFIT orany other sequence alignment program to determine whether a particularsequence is, for example, 95% identical to a reference sequenceaccording to the present invention, the parameters are set, of course,such that the percentage of identity is calculated over the full lengthof the reference polypeptide sequence and that gaps in homology of up to5% of the total number of amino acids in the reference sequence areallowed.

For purposes of the present invention, percent sequence identity may bedetermined using the Smith-Waterman homology search algorithm using anaffine gap search with a gap open penalty of 12 and a gap extensionpenalty of 2, BLOSUM matrix of 62. The Smith-Waterman homology searchalgorithm is taught in Smith and Waterman (1981) Adv. Appl. Math.2:482-489. A variant may, for example, differ from a referenceanti-SEMA4D antibody (e.g., MAb VX15/2503, 67 or 76) by as few as 1 to15 amino acid residues, as few as 1 to 10 amino acid residues, such as6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.

The constant region of an anti-SEMA4D antibody can be mutated to altereffector function in a number of ways. For example, see U.S. Pat. No.6,737,056B1 and U.S. Patent Application Publication No. 2004/0132101A1,which disclose Fc mutations that optimize antibody binding to Fcreceptors.

In certain anti-SEMA4D antibodies or fragments, variants or derivativesthereof useful in the methods provided herein, the Fc portion can bemutated to decrease effector function using techniques known in the art.For example, the deletion or inactivation (through point mutations orother means) of a constant region domain can reduce Fc receptor bindingof the circulating modified antibody thereby increasing tumorlocalization. In other cases, constant region modifications consistentwith the instant invention moderate complement binding and thus reducethe serum half-life. Yet other modifications of the constant region canbe used to modify disulfide linkages or oligosaccharide moieties thatallow for enhanced localization due to increased antigen specificity orantibody flexibility. The resulting physiological profile,bioavailability and other biochemical effects of the modifications, suchas tumor localization, biodistribution and serum half-life, can easilybe measured and quantified using well known immunological techniqueswithout undue experimentation. Anti-SEMA4D antibodies for use in themethods provided herein include derivatives that are modified, e.g., bythe covalent attachment of any type of molecule to the antibody suchthat covalent attachment does not prevent the antibody from specificallybinding to its cognate epitope. For example, but not by way oflimitation, the antibody derivatives include antibodies that have beenmodified, e.g., by glycosylation, acetylation, pegylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications can be carried outby known techniques, including, but not limited to specific chemicalcleavage, acetylation, formylation, etc. Additionally, the derivativecan contain one or more non-classical amino acids.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a side chain witha similar charge. Families of amino acid residues having side chainswith similar charges have been defined in the art. These familiesinclude amino acids with basic side chains (e.g., lysine, arginine,histidine), acidic side chains (e.g., aspartic acid, glutamic acid),uncharged polar side chains (e.g., glycine, asparagine, glutamine,serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g.,alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Alternatively, mutations can beintroduced randomly along all or part of the coding sequence, such as bysaturation mutaenesis, and the resultant mutants can be screened forbiological activity to identify mutants that retain activity (e.g., theability to bind an anti-SEMA4D polypeptide, to block SEMA4D interactionwith its receptor, or to decrease BBB permeability in a subject, e.g., apatient with a neuroinflammatory disorder).

For example, it is possible to introduce mutations only in frameworkregions or only in CDR regions of an antibody molecule. Introducedmutations can be silent or neutral missense mutations, i.e., have no, orlittle, effect on an antibody's ability to bind antigen. These types ofmutations can be useful to optimize codon usage, or improve ahybridoma's antibody production. Alternatively, non-neutral missensemutations may alter an antibody's ability to bind antigen. One of skillin the art would be able to design and test mutant molecules withdesired properties such as no alteration in antigen binding activity oralteration in binding activity (e.g., improvements in antigen bindingactivity or change in antibody specificity). Following mutagenesis, theencoded protein may routinely be expressed and the functional and/orbiological activity of the encoded protein, (e.g., ability toimmunospecifically bind at least one epitope of a SEMA4D polypeptide)can be determined using techniques described herein or by routinelymodifying techniques known in the art.

In certain embodiments, the anti-SEMA4D antibodies for use in themethods provided herein comprise at least one optimizedcomplementarity-determining region (CDR). By “optimized CDR” is intendedthat the CDR has been modified and optimized to improve binding affinityand/or anti-SEMA4D activity that is imparted to an anti-SEMA4D antibodycomprising the optimized CDR. “Anti-SEMA4D activity” or “SEMA4D blockingactivity” can include activity which modulates one or more of thefollowing activities associated with SEMA4D: B cell activation,aggregation and survival; CD40-induced proliferation and antibodyproduction; antibody response to T cell dependent antigens; T cell orother immune cell proliferation; dendritic cell maturation;demyelination and axonal degeneration; apoptosis of pluripotent neuralprecursors and/or oligodendrocytes; induction of endothelial cellmigration; inhibition of spontaneous monocyte migration; binding to cellsurface Plexin-B1 or other receptor, or any other activity associatedwith soluble SEMA4D or SEMA4D that is expressed on the surface ofSEMA4D+ cells. Anti-SEMA4D activity can also be attributed to a decreasein incidence or severity of diseases associated with SEMA4D expressionor overexpression, including, but not necessarily limited to,neuroinflammatory diseases including central nervous system (CNS) andperipheral nervous system (PNS) inflammatory diseases.

Examples of optimized antibodies based on murine anti-SEMA4D MAbs BD16and BB18, were described in US Publ. No. 2008/0219971 A1, InternationalPatent Application WO 93/14125 and Herold et al., Int. Immunol. 7(1):1-8 (1995), each of which are herein incorporated by reference in theirentirety. The modifications may involve replacement of amino acidresidues within the CDR such that an anti-SEMA4D antibody retainsspecificity for the SEMA4D antigen and has improved binding affinityand/or improved anti-SEMA4D activity.

V. Treatment Methods Using Therapeutic Anti-SEMA4D and Anti-PlexinB1Antibodies

Methods of the invention are directed to the use of an inhibitor ofSEMA4D interaction with a SEMA4D receptor, e.g., anti-SEMA4D bindingmolecules, anti-PlexinB1 binding molecules, or combination thereof,e.g., antibodies, including antigen-binding fragments, variants, andderivatives thereof, to decrease blood brain barrier permeability in asubject having a neuroinflammatory disorder. In certain embodiments, theneuroinflammatory disorder is, e.g., Multiple Sclerosis, AmyotrophicLateral Sclerosis, epilepsy, Alzheimer's Disease, Parkinson's Disease,meningitis, brain edema, brain trauma, or stroke. In certainembodiments, the endothelial cells express a SEMA4D receptor; and incertain embodiments, the receptor is Plexin-B1. Although the followingdiscussion refers to administration of an anti-SEMA4D antibody, ananti-PlexinB1 antibody, and combination thereof, the methods describedherein are also applicable to the antigen-binding fragments, variants,and derivatives of these anti-SEMA4D or anti-PlexinB1 antibodies thatretain the desired properties of the anti-SEMA4D or anti-PlexinB1antibodies of the invention, e.g., capable of specifically bindingSEMA4D, e.g., human, mouse, or human and mouse SEMA4D, having SEMA4Dneutralizing activity, and/or blocking the interaction of SEMA-4D withits receptor, e.g., Plexin-B1.

In one embodiment, treatment includes the application or administrationof an anti-SEMA4D binding molecule, an anti-PlexinB1 binding molecule,or combination thereof, e.g., an antibody or antigen binding fragmentthereof as described herein to a patient, where the patient has, or hasthe risk of developing a neuroinflammatory disorder. In anotherembodiment, treatment is also intended to include the application oradministration of a pharmaceutical composition comprising theanti-SEMA4D binding molecule, the anti-PlexinB1 binding molecule, orcombination thereof, e.g., an antibody or antigen binding fragmentthereof to a patient, where the patient has, or has the risk ofdeveloping a neuroinflammatory disorder. It should be appreciated thatdue to the interaction of SEMA4D with a receptor on endothelial cells,the application or administration of an anti-SEMA4D binding molecule, ananti-PlexitB1 binding molecule, or combination thereof is expected tooccur on the blood side of the blood brain barrier. By administering ananti-SEMA4D binding molecule, an anti-PlexinB1 binding molecules, orcombinations thereof by a route that exposes it to the blood side, e.g.including, but not limited to, intravenous administration, theanti-SEMA4D binding molecule, the anti-PlexinB1 binding molecules, orcombinations thereof will be permitted to inhibit the interaction ofSEMA4D with the SEMA4D receptor that is expressed by the endothelialcells.

The anti-SEMA4D binding molecules, anti-PlexinB1 binding molecules, orcombination thereof, e.g., antibodies or binding, fragments thereof asdescribed herein are useful for the treatment of variousneuroinflammatory disorders. In some embodiments, treatment of aneuroinflammatory disorder is intended to include a reduction, ordecrease, in permeability of the BBB. In other embodiments, treatment ofa neuroinflammatory disorder is intended to include an increase in theresistivity of the BBB. In other embodiments, treatment of aneuroinflammatory disorder is intended to include an increase in thenumber, density and/or concentration of endothelial cells present on theBBB. In other embodiments, treatment of a neuroinflammatory disorder isintended to include a change in the morphology or function orendothelial cells, or in the interactions among endothelial cells orastrocytes or between endothelial cells and astrocytes that form theBBB.

In one embodiment, the invention relates to the use of anti-SEMA4Dbinding molecules, anti-PlexinB1 binding molecules, or combinationthereof, e.g., antibodies or antigen-binding fragments, variants, orderivatives thereof, as a medicament, in particular for use in thetreatment or prophylaxis of neuroinflammatory disorders to inhibit,reduce, prevent, or minimize a breakdown in the BBB, or an increase inthe permeability of the BBB.

In accordance with the methods of the present invention, at least oneanti-SEMA4D binding molecule or anti-PlexinB1 binding molecule, e.g., anantibody or antigen binding fragment, variant, or derivative thereof, asdefined elsewhere herein can be used to promote a positive therapeuticresponse with respect to the neuroinflammatory disorder. A “positivetherapeutic response” with respect to the neuroinflammatory disorder isintended to include an improvement in the disease in association withthe anti-inflammatory activity, anti-apoptotic activity, or the like, ofthese antibodies, and/or an improvement in the symptoms associated withthe disease. That is, an anti-proliferative effect, the prevention offurther proliferation of the SEMA4D-expressing cell, a reduction in theinflammatory response including but not limited to reduced secretion ofinflammatory cytokines, adhesion molecules, proteases, immunoglobulins(in instances where the SEMA4D bearing cell is a B cell), combinationsthereof, and the like, increased production of anti-inflammatoryproteins, a reduction in the number of autoreactive cells, an increasein immune tolerance, inhibition of autoreactive cell survival, reductionin apoptosis, reduction in endothelial cell migration, increase inspontaneous monocyte migration, reduction in and/or a decrease in one ormore symptoms mediated by stimulation of sSEMA4D or SEMA4D-expressingcells can be observed. Such positive therapeutic responses are notlimited to the route of administration and may comprise administrationto the donor, the donor tissue (such as for example organ perfusion),the host, any combination thereof, and the like. In particular, themethods provided herein are directed to inhibiting, preventing,reducing, alleviating, or lessening the development of aneuroinflammatory disorder in a patient. Thus, for example, animprovement in the disease may be characterized as an absence ofclinically observable symptoms, a decrease in BBB permeability, anincrease in the number, density or concentration of endothelial cellspresent on the BBB, a change in the morphology or function of theendothelial cells, or a change in the interactions among endothelialcells and pericytes or astrocytes or between endothelial cells,pericytes and astrocytes that form the BBB.

Changes in the permeability of the BBB can be measured using in vitromodels. In certain embodiments, a dynamic in vitro DIV-BBB model can beemployed. Cucullo et al. have presented a DIV-BBB model composed ofnormal adult human brain microvascular endothelial cells and human adultastrocytes to study how haemodynamic changes and systemic inflammationaffect the integrity of the brain microvasculature. Specifically, thismodel uses a cartridge, or hollow tube, to represent the blood brainbarrier with the interior of the cartridge representing the blood sideof the blood brain barrier and the exterior of the cartridgerepresenting the brain side of the blood brain barrier. The interior ofthe cartridge is lined with adult human brain microvascular endothelialcells and exterior is lined with human adult astrocytes. As a bloodbrain barrier modifying agent, such as SEMA4D, is introduced into thelumen of the cartridge, the electrical current between the interior andexterior of the tube is monitored using Transendothelial ElectricalResistance Measurement, described below. One embodiment of this modelhas the novelty of having transcapillary microholes to enabletransendothelial cell trafficking between the vascular and theparenchymal compartment. An in depth description of the in vitro DIV-BBBmodel and the derivation and culture of the human microvascularendothelial cells and adult astrocytes employed can be found in, forinstance, Cucullo et al., Brain Research. 951 243-254 (2002); andCucullo et al., Journal of Cerebral Blood Flow & Metabolism. 2:767-77(2011). It should be appreciated that people skilled in the art willrecognize that other BBB models have been described and usefullyemployed for studies of the role of BBB in disease in the prior art andthat the present disclosure should not be limited to any one particularmodel.

The permeability of the BBB can be monitored using TransendothelialElectrical Resistance Measurement (TEER). TEER is used to monitor theintegrity of the BBB in real time, which has been shown to correlatewith the permeability of the BBB. The TEER system uses electronicmultiplexing to measure multiple cartridges in quick succession andassesses the integrity and viability of tissue culture bilayers rapidlyand reliably (Cucullo et al., 2002; Cucullo et al., 2010; Santaguida etal, 2006). In operation, the system applies an excitation voltage(0.06V) across the excitation electrodes inserted in each cartridge inthe luminal and extraluminal compartments. A microcontroller computesthe resistivity and capacitance (per cm²) of the barrier from physicalparameters. The values of capacitance are calculated by comparison ofthe voltage and current waveforms. The delay from peak-to-peak of thetwo waveforms is proportional to the capacitance value, which isexpressed as arch tension. The TEER can be measured from the initialsetup throughout the course of each experiment.

The anti-SEMA4D binding molecules, anti-PlexinB1 binding molecules, orcombination thereof, e.g., antibodies or antigen binding fragments,variants, or derivatives thereof can be used in combination with atleast one or more other treatments for neuroinflammatory disorders;where the additional therapy is administered prior to, during, orsubsequent to the anti-SEMA4D binding molecule, anti-PlexinB1 bindingmolecules, or combination thereof, e.g., antibody or antigen bindingfragment, variant, or derivative thereof, therapy. Thus, where thecombined therapies comprise administration of an anti-SEMA4D bindingmolecule, anti-PlexinB1 binding molecules, or combination thereof, e.g.,an antibody or antigen binding fragment, variant, or derivative thereof,in combination with administration of another therapeutic agent, themethods of the invention encompass coadministration, using separateformulations or a single pharmaceutical formulation, with simultaneousor consecutive administration in either order.

VI. Pharmaceutical Compositions and Administration Methods

Methods of preparing and administering anti-SEMMD binding molecules,anti-PlexinB1 binding molecules, or combination thereof, e.g.,antibodies, or antigen-binding fragments, variants, or derivativesthereof to a subject in need thereof are well known to or are readilydetermined by those skilled in the art. The route of administration ofthe anti-SEMA4D binding molecule, the anti-PlexinB1 binding molecule, orcombination thereof, e.g, antibody, or antigen-binding fragment,variant, or derivative thereof, can be, for example, oral, parenteral,by inhalation or topical. The term parenteral as used herein includes,e.g., intravenous, intraarterial, intraperitoneal, intramuscular,subcutaneous, rectal, or vaginal administration. While all these formsof administration are clearly contemplated as being within the scope ofthe invention, an example of a form for administration would be asolution for injection, in particular for intravenous or intraarterialinjection or drip. A suitable pharmaceutical composition for injectioncan comprise a buffer (e.g. acetate, phosphate or citrate buffer), asurfactant (e.g. polysorbate), optionally a stabilizer agent (e.g.albumin), etc. However, in other methods compatible with the teachingsherein, anti-SEMA4D binding molecules, anti-PlexinB1 binding molecules,or combination thereof, e.g., antibodies, or antigen-binding fragments,variants, or derivatives thereof can be delivered directly to the siteof the adverse cellular population thereby increasing the exposure ofthe diseased tissue to the therapeutic agent.

As discussed herein, anti-SEMA4D binding molecules, anti-PlexinB1binding molecules, or combination thereof, e.g., antibodies, orantigen-binding fragments, variants, or derivatives thereof can beadministered in a pharmaceutically effective amount for the in vivotreatment of neuroinflammatory disorders. In this regard, it will beappreciated that the disclosed binding molecules can be formulated so asto facilitate administration and promote stability of the active agent.In certain embodiments, pharmaceutical compositions in accordance withthe present invention comprise a pharmaceutically acceptable, non-toxic,sterile carrier such as physiological saline, non-toxic buffers,preservatives and the like. For the purposes of the instant application,a pharmaceutically effective amount of an anti-SEMA4D binding molecule,an anti-PlexinB1 binding molecule, or combination thereof, e.g., anantibody, or antigen-binding fragment, variant, or derivative thereof,shall be held to mean an amount sufficient to achieve effective bindingto a target and to achieve a benefit, e.g., to decrease the permeabilityof the BBB in a patient with a neuroinflammatory disorder.

The pharmaceutical compositions used in this invention comprisepharmaceutically acceptable carriers, including, e.g., ion exchangers,alumina, aluminum stearate, serum proteins, such as human serum albumin,buffer substances such as phosphates, glycine, sorbic acid, potassiumsorbate, partial glyceride mixtures of saturated vegetable fatty acids,water, salts or electrolytes, such as protamine sulfate, disodiumhydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zincsalts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone,cellulose-based substances, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol, andwool fat.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include, e.g., water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. In the subject invention, pharmaceutically acceptable carriersinclude, but are not limited to, 0.01-0.1 M and preferably 0.05 Mphosphate buffer or 0.8% saline. Other common parenteral vehiclesinclude sodium phosphate solutions, Ringer's dextrose, dextrose andsodium chloride, lactated Ringer's, or fixed oils. Intravenous vehiclesinclude fluid and nutrient replenishers, electrolyte replenishers, suchas those based on Ringer's dextrose, and the like. Preservatives andother additives may also be present such as, for example,antimicrobials, antioxidants, chelating agents, and inert gases and thelike.

More particularly, pharmaceutical compositions suitable for injectableuse include sterile aqueous solutions (where water soluble) ordispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersions. In such cases, thecomposition must be sterile and should be fluid to the extent that easysyringability exists. It should be stable under the conditions ofmanufacture and storage and will preferably be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquidpolyethylene glycol, and the like), and suitable mixtures thereof. Theproper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. Suitableformulations for use in the therapeutic methods disclosed herein aredescribed in Remington's Pharmaceutical Sciences (Mack Publishing Co.)16th ed. (1980).

Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride inthe composition. Prolonged absorption of the injectable compositions canbe brought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

In any case, sterile injectable solutions can be prepared byincorporating an active compound (e.g., an anti-SEMA4D antibody, orantigen-binding fragment, variant, or derivative thereof, by itself orin combination with other active agents) in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedherein, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the active compound into asterile vehicle, which contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-drying,which yields a powder of an active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.The preparations for injections are processed, filled into containerssuch as ampoules, bags, bottles, syringes or vials, and sealed underaseptic conditions according to methods known in the art. Further, thepreparations may be packaged and sold in the form of a kit. Sucharticles of manufacture can have labels or package inserts indicatingthat the associated compositions are useful for treating a subjectsuffering from, or predisposed to a disease or disorder.

Parenteral formulations can be a single bolus dose, an infusion or aloading bolus dose followed with a maintenance dose. These compositionscan be administered at specific fixed or variable intervals, e.g., oncea day, or on an “as needed” basis.

Certain pharmaceutical compositions used in this invention can be orallyadministered in an acceptable dosage form including, e.g., capsules,tablets, aqueous suspensions or solutions. Certain pharmaceuticalcompositions also can be administered by nasal aerosol or inhalation.Such compositions can be prepared as solutions in saline, employingbenzyl alcohol or other suitable preservatives, absorption promoters toenhance bioavailability, and/or other conventional solubilizing ordispersing agents.

The amount of an anti-SEMA4D binding molecule, an anti-PlexinB1 bindingmolecule, or combination thereof, e.g., antibody, or fragment, variant,or derivative thereof, to be combined with the carrier materials toproduce a single dosage form will vary depending upon the host treatedand the particular mode of administration. The composition can beadministered as a single dose, multiple doses or over an establishedperiod of time in an infusion. Dosage regimens also can be adjusted toprovide the optimum desired response (e.g., a therapeutic orprophylactic response).

In keeping with the scope of the present disclosure, anti-SEMA4Dantibodies, or antigen-binding fragments, variants, or derivativesthereof can be administered to a human or other animal in accordancewith the aforementioned methods of treatment in an amount sufficient toproduce a therapeutic effect. The anti-SEMA4D antibodies, orantigen-binding fragments, variants or derivatives thereof can beadministered to such human or other animal in a conventional dosage formprepared by combining the antibody of the invention with a conventionalpharmaceutically acceptable carrier or diluent according to knowntechniques. It will be recognized by one of skill in the art that theform and character of the pharmaceutically acceptable carrier or diluentis dictated by the amount of active ingredient with which it is to becombined, the route of administration and other well-known variables.Those skilled in the art will further appreciate that a cocktailcomprising one or more species of anti-SEMA4D binding molecules,anti-PlexinB1 binding molecules, or combinations thereof, e.g.,antibodies, or antigen-binding fragments, variants, or derivativesthereof, of the invention can be used.

By “therapeutically effective dose or amount” or “effective amount” isintended an amount of anti-SEMA4D binding molecule, anti-PlexinB1binding molecule, or combination thereof, e.g., antibody or antigenbinding fragment, variant, or derivative thereof, that when administeredbrings about a positive therapeutic response with respect to treatmentof a patient with a disease to be treated, e.g., a decrease in thepermeability of the BBB, an increase in the resistivity of the BBB, anincrease in the number, density or concentration of endothelial cellspresent on the BBB, a change in the morphology or function in theendothelial cells, or a change in the interactions among endothelialcells or astrocytes or between endothelial cells and astrocytes thatform the BBB.

Therapeutically effective doses of the compositions of the presentinvention, for the decrease in BBB permeability vary depending upon manydifferent factors, including means of administration, target site,physiological state of the patient, whether the patient is human or ananimal, other medications administered, and whether treatment isprophylactic or therapeutic. In certain embodiments the patient is ahuman, but non-human mammals including transgenic mammals can also betreated. Treatment dosages may be titrated using routine methods knownto those of skill in the art to optimize safety and efficacy.

The amount of at least one anti-SEMA4D binding molecule, anti-PlexinB1binding molecule, or combination thereof, e.g., antibody or bindingfragment, variant, or derivative thereof, to be administered is readilydetermined by one of ordinary skill in the art without undueexperimentation given the disclosure of the present invention. Factorsinfluencing the mode of administration and the respective amount of atleast one anti-SEMA4D binding molecule, anti-PlexinB1 binding molecule,or combination thereof, e.g., antibody, antigen-binding fragment,variant or derivative thereof include, but are not limited to, theseverity of the disease, the history of the disease, and the age,height, weight, health, and physical condition of the individualundergoing therapy. Similarly, the amount of anti-SEMA4D bindingmolecule, anti-PlexinB1 binding molecule, or combination thereof, e.g.,antibody, or fragment, variant, or derivative thereof, to beadministered will be dependent upon the mode of administration andwhether the subject will undergo a single dose or multiple doses of thisagent.

The invention also provides for the use of an anti-SEMA4D bindingmolecule, an anti-PlexinB1 binding molecule, or combination thereof,e.g., antibody of the invention, or antigen-binding fragment, variant,or derivative thereof, in the manufacture of a medicament for treating asubject for treating a neuroinflammatory disorder, wherein themedicament is used in a subject that has been pretreated with at leastone other therapy. By “pretreated” or “pretreatment” is intended thesubject has received one or more other therapies (e.g., been treatedwith at least one other neuroinflammatory therapy) prior to receivingthe Medicament comprising the anti-SEMA4D binding molecule, ananti-PlexinB1 binding molecule, or combination thereof, e.g., antibodyor antigen-binding fragment, variant, or derivative thereof.“Pretreated” or “pretreatment” includes subjects that have been treatedwith at least one other therapy within 2 years, within 18 months, within1 year, within 6 months, within 2 months, within 6 weeks, within 1month, within 4 weeks, within 3 weeks, within 2 weeks, within 1 week,within 6 days, within 5 days, within 4 days, within 3 days, within 2days, or even within 1 day prior to initiation of treatment with themedicament comprising the anti-SEMA4D binding molecule, for example, themonoclonal antibody VX15/2503 disclosed herein, or antigen-bindingfragment, variant, or derivative thereof. It is not necessary that thesubject was a responder to pretreatment with the prior therapy ortherapies. Thus, the subject that receives the medicament comprising theanti-SEMA4D binding molecule, an anti-PlexinB1 binding molecule, orcombination thereof, e.g., an antibody or antigen-binding fragment,variant, or derivative thereof could have responded, or could havefailed to respond, to pretreatment with the prior therapy, or to one ormore of the prior therapies where pretreatment comprised multipletherapies.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Such techniquesare explained fully in the literature. See, for example, Sambrook etal., ed. (1989) Molecular Cloning A Laboratory Manual (2nd ed.; ColdSpring Harbor Laboratory Press); Sambrook et al., ed. (1992) MolecularCloning: A Laboratory Manual, (Cold Springs Harbor Laboratory, NY); D.N. Glover ed., (1985) DNA Cloning, Volumes I and II; Gait, ed. (1984)Oligonucleotide Synthesis; Mullis et al. U.S. Pat. No. 4,683,195; Hamesand Higgins, eds. (1984) Nucleic Acid Hybridization; Hames and Higgins,eds. (1984) Transcription And Translation; Freshney (1987) Culture OfAnimal Cells (Alan R. Liss, Inc.); Immobilized Cells And Enzymes (IRLPress) (1986); Perbal (1984) A Practical Guide To Molecular Cloning; thetreatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Miller andCalos eds. (1987) Gene Transfer Vectors For Mammalian Cells, (ColdSpring Harbor Laboratory); Wu et al., eds., Methods In Enzymology, Vols.154 and 155; Mayer and Walker, eds. (1987) Immunochemical Methods InCell And Molecular Biology (Academic Press, London); Weir and Blackwell,eds., (1986) Handbook Of Experimental Immunology, Volumes I-IV;Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., (1986); and in Ausubel et al. (1989) CurrentProtocols in Molecular Biology (John Wiley and Sons, Baltimore, Md.).

General principles of antibody engineering are set forth in Borrebaeck,ed. (1995) Antibody Engineering (2nd ed.; Oxford Univ. Press). Generalprinciples of protein engineering are set forth in Rickwood et al., eds.(1995) Protein Engineering, A Practical Approach (IRL Press at OxfordUniv. Press, Oxford, Eng.). General principles of antibodies andantibody-hapten binding are set forth in: Nisonoff (1984) MolecularImmunology (2nd ed.; Sinauer Associates, Sunderland, Mass.); and Steward(1984) Antibodies, Their Structure and Function (Chapman and Hall, NewYork, N.Y.). Additionally, standard methods in immunology known in theart and not specifically described are generally followed as in CurrentProtocols in Immunology, John Wiley & Sons, New York; Stites et al.,eds. (1994) Basic and Clinical Immunology (8th ed; Appleton & Lange,Norwalk, Conn.) and Mishell and Shiigi (eds) (1980) Selected Methods inCellular Immunology (W.H. Freeman and Co., NY).

Standard reference works setting forth general principles of immunologyinclude Current Protocols in Immunology, John Wiley & Sons, New York;Klein (1982) J., Immunology: The Science of Self-Nonself Discrimination(John Wiley & Sons, NY); Kennett et al., eds. (1980) MonoclonalAntibodies, Hybridoma: A New Dimension in Biological Analyses (PlenumPress, NY); Campbell (1984) “Monoclonal Antibody Technology” inLaboratory Techniques in Biochemistry and Molecular Biology, ed. Burdenet al., (Elsevere, Amsterdam); Goldsby et al., eds. (2000) KubyImmunology (4th ed.; H. Freemand & Co.); Roitt et al. (2001) Immunology(6th ed.; London: Mosby); Abbas et al. (2005) Cellular and MolecularImmunology (5th ed.; Elsevier Health Sciences Division); Kontermann andDubel (2001) Antibody Engineering (Springer Verlan); Sambrook andRussell (2001) Molecular Cloning: A Laboratory Manual (Cold SpringHarbor Press); Lewin (2003) Genes VIII (Prentice Hall 2003); Harlow andLane (1988) Antibodies: A Laboratory Manual (Cold Spring Harbor Press);Dieffenbach and Dveksler (2003) PCR Primer (Cold Spring Harbor Press).

All of the references cited above, as well as all references citedherein, are incorporated herein by reference in their entireties.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLES

The following examples demonstrate the efficacy of anti-SEMA4D antibody(VX15/2503) in reducing or preventing the breakdown of the BBB, i.e., adecrease in permeability of the BBB, in an in vitro DIV-BBB model aswell as in an in vivo EAE model. An in vivo Alzheimer's Disease modelexperiment is also disclose herein. An in depth description about the invitro DIV-BBB model can be found in, for instance, Cucullo et al., BrainResearch. 951 243-254 (2002); and Cucullo et al., Journal of CerebralBlood Flow & Metabolism. 1-11 (2010). The in vivo EAE and Alzheimer'sDisease models are disclosed, e.g., in Miller et al., Curr ProtocImmunol. CHAPTER: Unit-15.1, 2007; Colton et al., J Alzheimers Dis15:571-587, 2008 and Wilcock et al., J. Neuroscience, 29:7957-7965,2009, respectively.

Example 1 Testing the Ability of an Anti-SEMA4D Binding Molecule, e.g.,an Antibody or Antigen-Binding Fragment, Variant, or Derivative Thereof,e.g., VX15/2503, to Restore the Integrity of the BBB FollowingSEMA4D-Induced Breakdown of the BBB in an In Vitro DIV-BBB ModelExperimental Design.

A dynamic in vitro BBB (“DIV-BBB”) model was performed to study theeffect of recombinant human SEMA4D (huSEMA4D-his) and VX15/2503(described in detail in US 2010/0285036 A1, incorporated herein byreference in its entirety) on the integrity of the BBB. Two DIV-BBBcartridges were tested in the model. The basic experimental design isshown in FIG. 1. Increasing concentrations of recombinant SEMA4D(rSEMA4D) were added into the lumen at 12 hour intervals, allowing forequilibration (approximately 12 hours/concentration). rSEMA4D wasinitially added into the lumen at a concentration of 0.05 μg/ml at time0. The concentration of rSEMA4D increased by 10-fold at each interval,for instance, 0.5 μg/ml at 12 hours, 5.0 μg/ml at 24 hours, and 50.0μg/ml at 36 hours. TEER measurements were taken between each interval asa reflection of changes in the permeability of the BBB at varyingconcentrations of rSEMA4D. Following addition of the final dose ofrSEMA4D at 50.0 μg/ml at 36 hours, VX15/2503 was added into the lumen ata concentration of 250 μg/ml at 48 hours. At 72 hours, 24 hoursfollowing the addition of VX15/2503, the permeability of the BBB wasagain measured.

Transendothelial Electrical Resistance Measurement (TEER) was used tomonitor the integrity of the BBB in real time. As mentioned above, theTEER system uses electronic multiplexing to measure multiple cartridgesin quick succession and assesses the integrity and viability of tissueculture bilayers rapidly and reliably (Cucullo et al., 2002; Santaguidaet al, 2006). In this dynamic in vitro model, the cartridges, or hollowtubes, were set up to represent the blood brain harrier with theinterior of the cartridge representing the blood side of the blood brainbarrier and the exterior of the cartridge representing the brain side ofthe blood brain barrier. The interior of the cartridge was lined withadult human brain microvascular endothelial cells and the exterior waslined with human adult astrocytes. As a blood brain barrier modifyingagent, such as SEMA4D, was introduced into the lumen of the cartridge,the electrical current between the interior and exterior of the tube wasmonitored using TEER. In operation, the TEER system applies anexcitation voltage (0.06V) across the excitation electrodes inserted ineach cartridge in the luminal and extraluminal compartments. Amicrocontroller computes the resistivity and capacitance (per cm²) ofthe barrier from physical parameters. The values of capacitance arecalculated by comparison of the voltage and current waveforms. The delayfrom peak-to-peak of the two waveforms is proportional to thecapacitance value, which is expressed as arch tension. The TEER wasmeasured from the initial setup throughout the course of eachexperiment.

rSEMA4D-Induced Increase in Permeability of the BBB.

Following formation of the BBB, the effect of rSEMA4D on the integrityof the BBB was measured by adding increasing concentrations ofrecombinant SEMA4D (rSEMA4D) into the lumen of the two cartridges.rSEMA4D was initially added into the lumen at a concentration of 0.05μg/ml at time 0. The concentration of rSEMA4D was increased 10-fold ateach 12 hour interval, for instance, 0.5 μg/ml at 12 hours, 5 μg/ml at24 hours and 50.0 μg/ml at 36 hours. TEER measurements were takenbetween and during each interval as a reflection of changes in thepermeability of the BBB at varying concentrations of rSEMA4D. Overall,permeability of the BBB remained relatively stable at 0.05 μg/ml ofrSEMA4D. Starting at 0.5 μg/ml, increasing concentrations of rSEMA4D(i.e., 0.5 μg/ml, 5 μg/ml and 50 μg/ml) resulted in decreased TEERmeasurement reflecting increased permeability of the endothelial celllayer. These results are shown in FIG. 2.

Antibody-Induced Decrease in Permeability of rSEMA4D Treated BBB.

To measure the effect of an anti-SEMA4D antibody on the BBB followingexposure to escalating dosage of rSEMA4D, VX15/2503 was added at aconcentration of 250 μg/ml at 48 hours. TEER measurements were taken at72 hours. Treatment with VX15/2503 resulted in an overall decrease inpermeability (or increase in resistivity) of the BBB in the twocartridges. This decrease in permeability reflects restoration of theBBB. The results are shown in FIG. 2.

Example 2 Testing the Ability of an Anti-SEMA4D Binding Molecule, e.g.,an Antibody or Antigen-Binding Fragment, Variant, or Derivative Thereof,e.g., VX15/2503, to Restore the Integrity of the BBB FollowingSEMA4D-Induced Breakdown of the BBB in an In Vitro DIV-BBB ModelExperimental Design.

A second experiment employing the in vitro DIV-BBB model was performedto study the effect of SEMA4D and VX15/2503 on the integrity of the BBB.The basic experimental design was similar to that shown in Example 1,and FIG. 1, above. For two weeks, the DIV-BBB cartridges underwent BBBformation in endothelial and astrocytic cell compartments. The formationof the BBB as reflected in TEER is shown in FIGS. 3 and 4.

rSEMA4D-Induced Increase in Permeability of the BBB.

Following formation of the BBB, the effect of rSEMA4D on the integrityof the BBB was measured by adding increasing concentrations ofrecombinant SEMA4D (rSEMA4D) into the lumen of the first cartridge of aset of three cartridges at 12 hour intervals, allowing for equilibration(approximately 12 hours/concentration). rSEMA4D was initially added intothe lumen at a concentration of 0.5 μg/ml at time 0. The concentrationof rSEMA4D increased by 10-fold at each interval, for instance, 5 μg/mlat 12 hours and 50.0 μg/ml at 24 hours. TEER measurements were takenbetween each interval as a reflection of changes in the permeability ofthe BBB at varying concentrations of rSEMA4D. Overall, increasingconcentrations of rSEMA4D resulted in decreased TEER measurementreflecting increased permeability of the BBB. These results are shown inFIG. 3.

To test the integrity of the BBB in the presence of an antigen that doesnot target the endothelial cell layer, a similarly prepared recombinantprotein control (CTRL, C35 protein) was added at equimolarconcentrations at the same 12 hour intervals (i.e., 0.25 μg/ml at time0, 2.5 μg/ml at 12 hours, and 25.0 μg/ml at 24 hours) to the twoadditional control cartridges. In contrast to the effect of rSEMA4D, theCTRL protein did not induce a significant change in TEER reflecting nomeaningful change in permeability of the BBB. If, however, 50.0 μg/ml ofrSEMA4D was added 12 hours after addition of the highest concentrationof CTRL protein, a rapid decrease in TEER similar to that observed withescalating doses of rSEMA4D was induced. The results are shown in FIG.4.

Antibody-Induced Decrease in Permeability of rSEMA4D Treated BBB.

Following addition of the final dose of rSEMA4D at 50.0 μg/ml at 24hours, the effect of VX15/2503 on TEER and the permeability of the BBBwas measured. In FIG. 3, VX15/2503 antibody was added at a concentrationof 250 μg/ml at 36 hours to two of the three cartridges that receivedescalating doses of rSEMA4D while the same concentration of an isotypecontrol antibody was added to the one remaining cartridge that hadreceived escalating doses of rSEMA4D. TEER measurements were taken atvarious subsequent points of time. Treatment with VX15/2503 resulted inan increase in TEER back to peak levels at the start of the experiment,reflecting an overall decrease in permeability of the BBB (i.e.,restoration of the BBB). In the one cartridge that received isotypecontrol antibody, TEER levels remained at the relatively reduced levelsinduced by treatment with rSEMA4D, indicating no meaningful decrease inpermeability of the BBB. Similar results are shown in FIG. 4. In FIG. 4,VX15/2503 antibody was added at a concentration of 250 μg/ml at 48 hoursto the two cartridges that received initial control recombinant C35protein followed by 50 μg/ml of rSEMA4D for 12 hours. Treatment withVX15/2503 resulted in an increase in TEER back to peak levels at thestart of the experiment., reflecting an overall decrease in permeabilityof the BBB (i.e., restoration of the BBB).

Example 3 Testing the Ability of an Anti-Plexin-B1 Binding Molecule,e.g., an Antibody or Antigen-Binding Fragment, Variant, or DerivativeThereof, to Restore the Integrity of the BBB Following SEMA4D-InducedBreakdown of the BBB in an In Vitro DIV-BBB Model

Another study was conducted to measure the effects of anti-Plexin-B1antibody (MAB37491 Human Plexin-B1 MAb (Clone 559830), R&D Systems) onthe integrity of the BBB. This antibody blocks binding of SEMA4D to thePlexin-B1 receptor. The results of this study are shown in FIG. 5. Asshown in FIG. 5, human endothelial cells and astrocytes in four DIV-BBBcartridges underwent BBB formation similar to the experiments describedabove. After BBB formation, rSEMA4D was added at a concentration of 50.0μg/ml, inducing an increase in BBB permeability (i.e., destruction ofthe BBB). Following addition of rSEMA4D, anti-Plexin-B1 antibody wasadded at a concentration of 125n/ml at 6 hours to two of the fourcartridges, VX15/2503 antibody was added at a concentration of 250 μg/mlto one of the four cartridges, and isotype control antibody was added ata concentration of 250n/ml to the remaining cartridge. TEER measurementswere taken at various subsequent points of time. Treatment with eitherVX15/2503 or anti-Plexin-B1 antibody resulted in an increase in TEERlevels with both agents. Treatment with VX15/2503 resulted in a somewhatgreater increase in TEER than treatment with anti-Plexin-B1 antibody atthe last time point. The effect of the two antibodies isindistinguishable at all other time points. The increase in TEERreflects an overall decrease in permeability of the BBB (i.e.,restoration of the BBB) in the presence of either VX15/2503 oranti-Plexin-B1 antibody. In the one cartridge that received isotypecontrol antibody, TEER levels remained at the relatively reduced levelsinduced by treatment with rSEMA4D, indicating no meaningful decrease inpermeability of the BBB. It should be appreciated that treatment canalso be conducted using a combination of VX15/2503 and anti-Plexin-B1.

Example 4 Testing the Ability of an Anti-SEMA4D Binding Molecule, e.g.,an Antibody or Antigen-Binding Fragment, Variant, or Derivative Thereof,e.g., VX15/2503, to Restore the Integrity of the BBB Following Breakdownof the BBB Induced by Activated PBMC and Flow Cessation in an In VitroDIV-BBB Model Experimental Design.

Another experiment employing the in vitro DIV-BBB model was performed tostudy the effect of VX15/2503 on restoring the integrity of the BBBfollowing breakdown of the BBB induced by activated peripheral bloodmononuclear cells (PBMC) and flow cessation. For two weeks, two DIV-BBBcartridges underwent BBB formation in endothelial and astrocytic cellcompartments.

Activated PBMC-Induced Increase in Permeability of the BBB.

Following formation of the BBB, the effect of activated PBMC on theintegrity of the BBB was measured. PBMC were activated withPMA/ionomycin for 2 hours and then added at a concentration of 10⁶/mlinto the lumen of the two cartridges. TEER measurements were taken priorto and after the addition of the activated PBMC as a reflection ofchanges in the permeability of the BBB. Overall, adding activated PBMCto the cartridges at 10⁶/ml resulted in decreased TEER measurementreflecting increased permeability of the BBB. These results are shown inFIG. 6.

At approximately 2-4 hours following the addition of the activated PBMCto the cartridges, flow cessation was performed for 1 hour. TEERmeasurements were taken before and after flow cessation as a reflectionof changes in the permeability of the BBB. Overall, flow cessationresulted in a further decrease in TEER measurement reflecting increasedpermeability of the BBB. These results are also shown in FIG. 6.

Antibody-Induced Decrease in Permeability of the BBB Exposed toActivated PBMC.

Following exposure to activated PBMC and flow cessation, the effect ofVX15/2503 on TEER and the permeability of the BBB was measured.VX15/2503 antibody was added at a concentration of 250 μg/ml to one ofthe two cartridges that received activated PBMC while the sameconcentration of an isotype control antibody (Isotype Control Ig, 2269)was added to the remaining cartridge. TEER measurements were taken atvarious subsequent points of time. As shown in FIG. 6, treatment withVX15/2503 resulted in an increase in TEER back to peak levels at thestart of the experiment, reflecting an overall decrease in permeabilityof the BBB (i.e., restoration of the BBB). In the cartridge thatreceived isotype control antibody, TEER levels remained at therelatively reduced levels induced by treatment with activated PBMC andflow cessation, indicating no meaningful decrease in the permeability ofthe BBB.

Example 5

Testing the Ability of an Anti-SEMA4D Binding Molecule, e.g., anAntibody or Antigen-Binding Fragment, Variant, or Derivative Thereof,e.g., VX15/2503, to Protect the Integrity of the BBB in an In Vivo EAEModel

Anti-SEMA41) binding molecules, e.g., antibodies or antigen-bindingfragments, variants or derivatives thereof, e.g., VX15/2503, were testedin the in vivo experimental autoimmune encephalomyelitis (EAE) model.

In an in vivo EAE model, the breakdown of the BBB was investigated byexamining changes in brain permeability as reflected in the penetrationof fibrinogen from blood into the brain parenchyma and throughexamination of endothelial tight junction proteins, including Claudin-5.In this model, EAE was induced in mice by immunization with PLP peptide(139-151). Of course, those skilled in the art will appreciate thatother EAE inducing proteins may be used as well (e.g., a myelin antigen,for instance myelin oligodendrocyte glycoprotein peptide 35-55) andthat, for greatest efficiency, these inducing proteins or peptides mayvary from one species to another and from one strain of mice to another,Steinman, L. Neuron 24:511-514 (1999). Tissue sections from the centralnervous system (CNS) of animals at different stages of disease were thenimmunostained for proteins (fibrinogen and claudin-5, which serve asmarkers for BBB disruption).

Experimental Design.

In an in vivo EAE model, EAE was induced in 12 week old SJL/J mice (10mice per group) by immunization with PLP peptide (139-151) in CFA(complete Freund's adjuvant). The mice were then treated once per weekfrom 7 days post-induction with 600 μg anti-SEMA4D antibody (VX15/2503antibody) or control IgG. Neurological signs were first observed at 11dpostinduction (dpi). At 13 days postinduction, during the acute phase ofdisease, 4 mice per group were sacrificed and lumbar spinal cord sampleswere prepared for histopathologic analysis. To detect BBB disruption inthe samples, these samples were immunostained for fibrinogen andclaudin-5. The procedure for immunostaining is as follows: Sections wererinsed twice in PBS, then incubated in PBS 0.1% glycine 10 min, blockedin PBS 0.3% Triton X-100 10% goat serum for 1 h, and incubated withprimary Abs in blocking buffer overnight at 4° C. For claudin-5 (CLN-5),prior to blocking, sections were soaked in EDTA, pH 8, 100° C. Primaryantibodies used were anti-CLN-5 (1:50), and anti-fibrinogen (1:1,000).After washing three times in PBS 0.3%, Triton X-100 sections were thenincubated in relevant species specific secondary antibodies conjugatedto AlexaFluor 488 and/or AlexaFluor 594 (1/100; Molecular Probes) inblocking buffer for 1 h at 25° C., washed again three times, andcounterstained with 4,6-diamidino-2-phenylindole (DAPI). All sampleswere examined and photographed using a Zeiss LSM 510 META laser scanningconfocal system attached to an Axiovert 200 inverted fluorescencemicroscope.

Clinical disease in the mice was scored as follows: 0=no symptoms;1=floppy tail; 2=hind limb weakness; 4=fore and hind limb weakness;5=death. Neurological signs were first observed at 11 dayspostinduction. In the mice treated with the VX15/2503 antibody, clinicaldisease reached a mean severity score of 0.75, indicative of mild tailweakness, while clinical disease in mice of the control group reached amean severity scope of 2.25, indicative of paraparesis.

Results of the immunostaining at 13 days postinduction are shown inFIGS. 7A-7C. Fibrinogen does not normally penetrate the blood-brainbrain barrier (BBB). In EAE, with the BBB compromised, the greenfibrinogen stain was detected in brain matter (left panel). In addition,expression of claudin-5 (CLN-5, red stain), a component of the tightjunctions that make up the BBB, was reduced. Mice in the control groupshowed reduced expression of claudin-5 and increased levels ofextravascular leakage of fibrinogen, which correlated with a disruptionin the BBB. In mice treated with VX15/2503 antibody, on the other hand,expression of claudin-5 was maintained and leakage of fibrinogen wassignificantly reduced. These results demonstrated the protective effectof VX15/2503 antibody against disruption of the BBB in these treatedmice, and specifically demonstrated how anti-SEMA4D antibody preventsBBB breakdown, prevents extravascular leakage of fibrinogen (7A leftpanel and quantitation in 7B), and preserves claudin-5 as detected byred stain (7 A right panel and quantitation in 7C).

Example 6 Effect of SEMA4D on Tight Junction Proteins in Cultures ofCerebral Endothelial Cells Experimental Design.

The expression of the key endothelial tight junction protein Claudin-5following treatment of CNS derived endothelial cells with solublerecombinant SEMA4D was investigated. In this model, primary mousecentral nervous system (CNS) endothelial cultures were isolated andplated on a 6-well matrigel-coated plate (isolated MBCEC from 10 brainswere resuspended in 3 ml primary endothelial cell culture medium andplated at 250 ul per well). Cultures were used at day 7 after isolation.Cultures were treated with 1 ng/ml, 10 ng/ml or 100 ng/ml recombinantmouse SEMA4D or 100 ng/ml mouse VEGF-A (positive control) for 24 hours.The endothelial cultures of the animals were then subjected toSDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting forthe claudin-5 tight junction protein and actin loading control. Datawere scanned and subjected to densitometry using ImageJ software (NIH).

Results of immunoblotting are shown in FIG. 8. As provided in FIG. 8,endothelial cell cultures treated with 100 ng/ml of recombinant SEMA4Dshowed a significant reduction in Claudin-5 protein expression.Endothelial cell cultures treated with 100 ng/ml of VEGF-A were testedas a positive control for down-regulation of Claudin-5. Thisdemonstrates the important role of SEMA4D in regulating expression of akey tight junction protein of the BBB.

Example 7 Testing the Ability of an Anti-SEMA4D or Anti-PlexinB1 BindingMolecule, e.g., an Antibody or Antigen-Binding Fragment, Variant, orDerivative Thereof to Decrease the Permeability of the BBB in an In VivoAlzheimer's Disease (AD) Model

Anti-SEMA4D or Anti-PlexinB1 binding molecules, e.g., antibodies orantigen-binding fragments, variants or derivatives thereof, e.g., MAb 67(described in detail in US 2010/0285036 A1, incorporated herein byreference in its entirety), are tested in various model systems ofneuroinflammatory disorders, including, but not limited to an in vivoexperimental Alzheimer's disease (AD) transgenic mouse modelAPPSwDI/NOSC^(−/−). These mice were generated by crossingAPP-Swedish-Dutch-Iowa mutant mouse with nitric oxide synthase 2knock-out mouse (Colton et al., J Alzheimers Dis. 15:571-587, 2008; VanNostrand et al., Stroke 41:S135-S138, 2010). APPSwDI/NOSC^(−/−) micedevelop age-related neurovascular amyloidosis with disrupted BBBfunction, intraparenchymal amyloid plaques, mouse tauhyperphosphorylation, neuroinflammation, neuronal cell death, andcognitive deficits. Wilcock et al. have shown that treatment ofAPPSwDI/NOSC^(−/−) mice with amyloid-beta directed active immunotherapyleads to marked reduction in amyloid deposition, but with increasedincidence of microhemorrhages (Wilcock et al., J Neurosci. 29:7957-7965,2009).

In an in vivo AD model, the progression of AD is investigated byexamining immunohistochemical signatures of amyloid deposition, tauhyperphosphorylation, and BBB leakage (fibrinogen), as well as byassessing cognitive abilities in spatial memory-based behavioralparadigms. In this model, the transgenic mice are administered MAb 67 orControl Ig (Mab 2B8) intravenously at a concentration of 30 mg/kg fromage 26 to 38 weeks for a total of 13 doses.

The mice are initially subjected to baseline behavioral testing at age10-12 weeks, e.g., Open field, RAWn and Barnes Maze tests, and micereaching the criteria of activity and learning/memory are included inthe follow-up. Behavioral deficits are again measured at age 38, 39 and40 weeks and body weight is recorded. Mice that do not reach criteriafor study enrollment will be sacrificed. At the 41 week of ageend-point, the animals will be euthanized and the brains will beprocessed for biochemical and immunohistological analyses for solubleand insoluble amyloid beta levels and deposits. Serum is collectedpre-dosing, during dosing and at the end-point for PK at age 10, 25 and41 weeks. Tissue sections from the central nervous system (CNS) ofanimals at different stages of disease may be immunostained forfibrinogen, that can be used as markers for BBB disruption.

Many modifications and other embodiments of the inventions set forthherein will come to mind to one skilled in the art to which theseinventions pertain having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the inventions are not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims and listof embodiments disclosed herein. Although specific terms are employedherein, they are used in a generic and descriptive sense only and notfor purposes of limitation.

1. A method of decreasing blood brain barrier permeability in a subjecthaving a neuroinflammatory disorder, comprising administering to thesubject an effective amount of an isolated binding molecule whichspecifically binds to semaphorin-4D (SEMA4D), thereby decreasing bloodbrain barrier permeability in the subject.
 2. The method of claim 1,wherein the binding molecule maintains or increases Claudin-5 expressionin the subject.
 3. (canceled)
 4. The method, of, claim 1, wherein thebinding molecule inhibits semaphorin 4D (SEMA4D) interaction with aSEMA4D receptor.
 5. (canceled)
 6. The method of claim 4, wherein theSEMA4D receptor is Plexin-B1.
 7. The method of claim 4, wherein thebinding molecule specifically binds to SEMA4D or Plexin-B1.
 8. Themethod of any one of claim 1, 2, 4, 6, 7 or 21, wherein the bindingmolecule competitively inhibits a reference monoclonal antibody selectedfrom the group consisting of VX15/2503 or 67 from specifically bindingto SEMA4D.
 9. A method of decreasing blood-brain barrier permeability ina subject having a neuroinflammatory disorder, comprising administeringto the subject an effective amount of an isolated binding molecule whichspecifically binds to SEMA4D, wherein the binding molecule competitivelyinhibits a reference monoclonal antibody selected from the groupconsisting of VX15/2503 or 67 from specifically binding to SEMA4D,thereby decreasing blood brain barrier permeability in the subject. 10.The method of claim 1 or 9, wherein the binding molecule specificallyhinds to the same SEMA4D epitope as a reference monoclonal antibodyselected from the group consisting of VX15/2503 or
 67. 11. The method ofclaim 1 or 9, wherein the binding molecule comprises an antibody orantigen-binding fragment thereof.
 12. The method of claim 11, whereinthe antibody or antigen-binding fragment thereof comprises a variableheavy chain (VH) comprising VHCDRs 1-3 comprising SEQ ID NOs 6, 7, and8, respectively, and a variable light chain (VL) comprising VLCDRs 1-3comprising SEQ ID NOs 14, 15, and 16, respectively.
 13. The method ofclaim 12, wherein the antibody or antigen-binding fragment thereof ismonoclonal antibody VX15/2503 or
 67. 14. The method of claim 7, whereinthe binding molecule specifically binds to Plexin-B1.
 15. The method ofclaim 14, wherein the binding molecule competitively inhibits SEMA4Dfrom binding to Plexin-B1.
 16. The method of claim 14 or 15, wherein thebinding molecule is an anti-Plexin-B1 antibody or antigen bindingfragment thereof.
 17. (canceled)
 18. A method of treating a subjecthaving a neuroinflammatory disorder, comprising administering to thesubject an effective amount of an inhibitor of semaphorin 4D (SEMA4D)interaction with a SEMA4D receptor, wherein the inhibitor decreasespermeability of the blood-brain barrier, thereby treating the subject.19. The method of claim 18, wherein the inhibitor is selected from thegroup consisting of an anti-SEMA4D binding molecule, an anti-PlexinB1binding molecule, a small molecule inhibitor of SEMA4D, or a smallmolecule inhibitor of a SEMA4D receptor.
 20. The method of any one ofclaim 1, 9, or 18, wherein the neuroinflammatory disorder is selectedfrom the group consisting of Multiple Sclerosis, Amyotrophic LateralSclerosis, epilepsy, Alzheimer's Disease, Parkinson's Disease,meningitis, brain edema, and brain trauma.
 21. The method of claim 4 or6, wherein the binding molecule specifically binds to SEMA4D andPlexin-B1.